Polypeptides targeting vascular endothelial growth factor receptor-2 and αvβ3 integrin

ABSTRACT

Polypeptides comprising variant vascular endothelial growth factor sequences are provided. The polypeptides are useful in cancer imaging, cancer diagnosis, monitoring and treatment as well as treatment of diseases characterized by excessive neovascularization.

FIELD OF THE INVENTION

The present invention relates to the field of angiogenesis-relateddiseases and their diagnosis, characterization and treatment.

BACKGROUND OF THE INVENTION

Angiogenesis, the process of new blood vessel formation from preexistingvasculature, plays critical roles in both normal physiological processessuch as wound healing, pregnancy, tissue regeneration and in thepathogenesis of cancer, rheumatoid arthritis, and diabetic microvasculardisease (see Carmeliet P (2005), Nature 438, pp. 932-936), and isregulated by a large number of pro- and antiangiogenic cytokines andgrowth factors (Ferrara N (2000), Curr Opin Biotechnol 11, pp. 617-624).During adulthood, most blood vessels remain quiescent and angiogenesisoccurs only in the cycling ovary and in the placenta during pregnancy.

However, when angiogenic growth factors are produced in excess ofangiogenesis inhibitors, endothelial cells are stimulated toproliferate. A number of angiogenic growth factors have been describedto date among which vascular endothelial growth factor (VEGF) appears toplay a key role as a positive regulator of physiological andpathological angiogenesis (Brown et al. (1997) in “Control ofAngiogenesis” (Goldberg and Rosen, eds.), Birkhauser, Basel, pp.233-269; Thomas K A (1996), J Biol Chem 271, pp. 603-606; Neufeld et al.(1999), FASEB J 13, pp. 9-22).

The focus on inhibition of angiogenesis for treatment of cancer andmacular degeneration has largely focused on targeting vascularendothelial growth factor (VEGF) and its receptors due to the prominentrole of this pathway in vascular formation. VEGF-mediated signaling ismediated through its interactions with two receptor tyrosine kinases,VEGFR1 (Flt-1) and VEGFR2 (Flk-1 or KDR). VEGFR2, which is expressed invascular endothelial cells, monocytes, macrophages, and hematopoieticstem cells, is the primary mediator of the mitogenic and angiogeniceffects of VEGF. VEGF is a homodimeric ligand that binds two moleculesof VEGFR2, one at each pole, thereby triggering receptor dimerizationand activation, with a K_(D) of around 100 pM. The role of VEGFR1 isless clear, but it appears to function as a ‘decoy’ receptor thatnegatively regulated VEGF signaling by preventing VEGF from bindingVEGFR2. VEGF-A is the main ligand for VEGFR2, but proteolyticallycleaved forms of VEGF-C and VEGF-D may also bind to and activate VEGFR2.Hence, it may be beneficial to target VEGFR2 directly in order to bestinhibit angiogenic processes.

Integrins are a diverse class of heterodimeric (α/β) receptors involvedin cell adhesion to extracellular matrix ligands. In particular,integrin αvβ3 has been implicated as critically involved in tumorproliferation, metastasis, and angiogenesis, and there have thereforebeen many efforts to develop anti-cancer therapies that target integrinαvβ3. Interestingly, there may be a critical link between integrin αvβ3and VEGF2-stimulated angiogenesis. Moreover, cross-talk and synergyexists between integrins and growth factor receptors. In particular,engagement of αvβ3 integrin on endothelial cells promotesphosphorylation and activation of VEGFR2, thereby augmenting themitogenic activity of VEGF. It has been shown that β33 binds to VEGFR2to potentiate its activity, and that αvβ3 antagonists decrease theβ3-VEGFR2 interactions and VEGFR2 activation (though not VEGFR2expression levels). These studies suggest that VEGFR2-mediatedangiogenesis is potentiated by integrin αvβ3.

Numerous other factors are involved in angiogenic processes, includingtransforming growth factors alpha and beta (TGF-α and -β), tumornecrosis factor (TNF), and fibroblast growth factor (FGF). Accordingly,blocking of single angiogenic molecules may have only modest effect onslowing tumor growth because there multiple angiogenesis pathways thatcan replace VEGF as the cancer progresses. Thus, there has beenconsiderable interest in developing biological agents capable of bindingto more than one set of ligand-receptor interactions in order to moreefficiently block angiogenic processes.

Publications

Siemeister et al. (1998) “An antagonistic vascular endothelial growthfactor (VEGF) variant inhibits VEGF-stimulated receptorautophosphorylation and proliferation of human endothelial cells”, ProcNatl Acad Sci USA 95, pp. 4625-4629 and Boesen et al. (2002)“Single-chain vascular endothelial growth factor variant with antagonistactivity”, J Biol Chem 277 (43), pp. 40335-40341, disclose thepreparation of a single-chain VEGF variants.

WO02081520 by Thomas P. Boesen and Torben Halkier, filed Apr. 8, 2002,and entitled “Single Chain Dimeric Polypeptides”, discloses asingle-chain dimeric polypeptide which binds to an extracellularligand-binding domain of VEGFR2 or VEGFR3 receptor and which functionsas a receptor antagonist for prevention or treatment of a disease orcondition involving increased signal transduction from or increasedactivation of the VEGFR2 and/or VEGFR3 receptor, e.g. to inhibitangiogenesis or lymphangiogenesis. See also Ferrara et al. (2003) NatureMedicine 9:669-676; Ferrara and Kerbal. (2005) Nature 438:967-974; Meyeret al. (2006) Current Pharmaceutical Design 12:2723-2747; Silverman etal. (2009) Journal of Molecular Biology 385:1064-1075; Richards et al.(2003) Journal of Molecular Biology 326:1475-1488; Boesen et al. (1998)Proceedings of the National Academy of Sciences 95:4625-4629; Kiba etal. (2003) Journal of Biological Chemistry 278:13453-13461.

SUMMARY OF THE INVENTION

Compositions are provided of single-chain antagonistic human VEGFvariants. The single-chain VEGF variants of the invention bind to VEGFreceptors, including VEGFR2 receptors, but do not induce receptoractivation, thereby antagonizing VEGF-stimulated receptorautophosphorylation and proliferation of endothelial cells. Compositionsinclude the polypeptide or polypeptides of the invention, which may beprovided as a single species or as a cocktail of two or morepolypeptides, usually in combination with a pharmaceutically acceptableexcipient. Compositions also include nucleic acids encoding suchpolypeptides. In some embodiments the polypeptide of the invention isconjugated to a functional moiety, e.g. a detectable label such afluorescent label, a detectable label such as an isotopic label; acytotoxic moiety, and the like, which may find use in imaging,quantitation, therapeutic purposes, etc.

In some embodiments the polypeptide of the invention is a single-chainantagonistic human VEGF variant having increased affinity for theVEGF2R, relative to the native polypeptide. Such polypeptides includewithout limitation those set forth in SEQ ID NO:9-18.

In some embodiments the polypeptide of the invention is a bifunctionalsingle-chain antagonistic human VEGF variant comprising a native VEGFsequence, an amino acid linker, and a modified VEGF, where the modifiedVEGF comprises a loop with an integrin-recognition RGD sequence capableof binding αvβ3 integrin. Such polypeptides include without limitationthose set forth in SEQ ID NO:5-8. Such polypeptides also include anypolypeptide of SEQ ID NO:9-18 and 19-27, further comprising themodification of replacing amino acid residues of loop 2 or loop 3 in themutated VEGF pole with an RGD motif, which RGD motif includes, withoutlimitation XXRGDXXXX, XXXRGDXXX, or XXXXRGDXX. Specific RGD motifs ofinterest include those set forth in SEQ ID NO:29-SEQ ID NO:75. The RGDmotif may be screened for binding to an αvβ3 integrin, an αvβ5 integrin,an α5β1 integrin, etc. In some embodiments the loop 3 sequence (SEQ IDNO:76) IKPHQGQ is replaced with the RGD motif. In other embodiments amotif for binding to a vascular protein other than αvβ3 integrin isprovided in the scVEGF.

In some embodiments the polypeptide of the invention is a bifunctionalsingle-chain antagonistic human VEGF variant having increased affinityfor the VEGF2R, relative to the native polypeptide. Such polypeptidesinclude without limitation those set forth in SEQ ID NO:19-27.

Methods are provided that utilize the polypeptides of the invention forimaging normal tissue, abnormal tissue, precancerous tissue, cancer, andtumors. In other embodiments methods are provided for diagnosis ofprecancerous tissue, cancer, and tumors. In other embodiments thebifunctional single-chain antagonistic VEGF variant of the invention isused in the treatment of an individual having a vascularized tumor orcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate embodiments of the invention and,together with the description, serve to explain the invention. Thesedrawings are offered by way of illustration and not by way oflimitation.

FIG. 1. Design of scVEGF and binding of yeast-displayed wt and mutvariants. (A) Structure of VEGF. Chains 1 and 2 are shown in dark blueand light blue, respectively. Mutations in scVEGFmut are shown in red.(B) Binding of scVEGFwt (●) and scVEGFmut (▪) to recombinant humanVEGFR2 extracellular domain. (C) Loops subjected to saturationmutagenesis in scVEGFmut are shown. Loop 1, pink; loop 2, green; loop 3,red. Since it is difficult to determine which residues constitute thestart and end of these loops (only loop 1 is disulfide constrained),multiple different amino acid registers were substituted for each loop.

FIG. 2. Comparison of VEGF-VEGFR2 interaction with the variants proposedin this study. (A) Wild-type VEGF is a homodimer that binds to twoVEGFR2 molecules to activate signaling. (B) Single-chain VEGF has one ofthe VEGFR2 binding sites mutated, preventing a second receptor moleculefrom binding and thereby antagonizing signaling. (C) The bispecificvariants engineered here have one VEGFR binding site mutated with anαvβ3 integrin recognition loop, making them capable of binding VEGFR2and/or αvβ3 integrin.

FIG. 3. FACS plots showing sorting of scVEGFrgd loop libraries. (A) Sortround 1, 250 nM α_(v)β₃ integrin. (B) Sort round 4, 100 nM VEGFR2-Fc.(C) After sort round 7, 25 nM α_(v)β₃ integrin.

FIG. 4. FACS plots showing sorting of scVEGFmut (A-D) and scVEGFrgd-7B(E-H) error-prone mutagenesis libraries.

FIG. 5. VEGF structure showing positions of the most common mutationsselected from the scVEGF affinity maturation libraries. Chains 1 and 2are shown in dark and light blue, respectively. Mutations in scVEGFmutgeared toward disrupting binding at one pole of the protein are shown inred. Mutations commonly appearing in the affinity matured clones areshown in green.

FIG. 6. SPR interaction analysis of scVEGF variants with VEGFR2.Representative tracing of association and dissociation of differentconcentrations of scVEGF variants, during perfusion at 30 μl/min overVEGFR-2 immobilized to CM-5 sensor chip.

FIG. 7. Binding titrations against cell lines. (A) K562α_(v)β₃ cells.(B) PAE cells (C) PAE-KDR cells (also express porcine α_(v)β₃). (D)HUVEC cells (E) U87MG cells (F) SVR cells.

FIG. 8. Effect of scVEGF variants on VEGF-stimulated tyrosinephosphorylation of VEGFR2 in HUVEC in the absence (A) or presence (B) ofvitronectin. Results from vitronectin-free (C-D) and experimentsincluding vitronectin (E) were analyzed by densitometry on chemidoc andvalues are means of three independent experiments. Bars, ±SD.

FIG. 9. Effect of scVEGF variants on VEGF-stimulated HUVEC proliferationin the absence (A-B) or presence (C-D) of vitronectin. Values expressedas means of three independent experiments. Bars, ±SD.

FIG. 10. Inhibition of vitronectin-mediated HUVEC adhesion by engineeredscVEGF variants. Vitronectin-coated wells were incubated with cells for2 h with the indicated concentrations of scVEGF variants. Adherent cellsremaining after several wash steps were quantified by crystal violetstaining and determining the absorbance at 600 nm. Values werebackground subtracted using a negative control containing no peptide.Symbols scVEGF variants (×) scVEGFwt; (▪) scVEGF-7H; (♦) scVEGF-7I; (●)scVEGFmut; (▴) scVEGF-7P.

FIG. 11. Testing scVEGFmut loop libraries for protein expression andbinding to VEGFR2.

FIG. 12. Testing scVEGFmut and scVEGFrgd-7B affinity matured clones forbinding to VEFGR2.

DETAILED DESCRIPTION OF THE EMBODIMENTS

VEGFR2 and αvβ3 integrin are critical effectors of tumor angiogenesiswith broad clinical utility for the early detection of many solidcancers. Therapeutic and diagnostic agents that selectively inhibit orantagonize VEGFR2 as well as αvβ3 integrin are beneficial for treatingangiogenesis-related disorders, in particular neoplasia and tumormetastasis. In addition to cancer, other proliferative diseasescharacterized by excessive neovascularization, e.g. psoriasis,age-related macular degeneration, diabetic retinopathy, rheumatoidarthritis, and the like, are treated with an effective dose ofpolypeptides of the invention, where the dose is effective at inhibitingangiogenesis.

DEFINITIONS

“VEGF” is a secreted disulfide-linked homodimer that selectivelystimulates endothelial cells to proliferate, migrate, and producematrix-degrading enzymes, all of which are processes required for theformation of new vessels. In addition to being the only knownendothelial cell specific mitogen, VEGF is unique among angiogenicgrowth factors in its ability to induce a transient increase in bloodvessel permeability to macromolecules. The term “VEGF” as used hereinrefers to proteins that are also known in the literature as “VEGF-A”,i.e. the VEGF isoforms containing 121, 145, 165, 189 or 206 amino acidresidues as described herein, in contrast to “VEGF-C” and “VEGF-D”.

The human VEGF gene is organized in eight exons, separated by sevenintrons. Alternative exon splicing of the VEGF gene results in thegeneration of at least five different molecular species, havingrespectively 121, 145, 165, 189 and 206 amino acids (VEGF-121, VEGF-145,VEGF-165, VEGF-189, VEGF-206); these isoforms differ not only in theirmolecular weight but also in their biological properties, such as theability to bind to cell surface heparin sulfate proteoglycans. VEGF-165is the predominant molecular species produced by a variety of normal andtransformed cells (Houck et al. (1991), Mol Endocrinol 5, pp. 1806-1814;Carmeliet et al. (1999), Nature Med 5, pp. 495-502).

VEGF signaling is mediated largely via two homologous,endothelium-specific tyrosine kinase receptors, VEGFR1 (Flt-1 akafms-like tyrosine kinase 1) and VEGFR2 (Flk-1/KDR aka kinase domainreceptor) whose expression is highly restricted to cells of endothelialorigin (de Vries et al. (1992), Science 255, pp. 989-991; Millauer etal. (1993), Cell 72, pp. 835-846; Terman et al. (1991), Oncogene 6, pp.519-524). Both receptors have an extracellular domain consisting ofseven IgG-like domains, a transmembrane domain and an intracellulartyrosine kinase domain. The affinity of VEGFR1 for VEGF (Kd=1-20 pM) ishigher compared to that of VEGFR2 (Kd=50-770 pM) (Brown et al. (1997) in“Control of Angiogenesis” (Goldberg and Rosen, eds.), Birkhauser, Basel,pp. 233-269; de Vries et al. (1992), Science 255, pp. 989-991; Terman etal. (1992), Biochem Biophys Res Commun 187, pp. 1579-1586).

While VEGFR1 is essential for physiologic and developmentalangiogenesis, VEGFR2 is the major mediator of the mitogenic, angiogenicand permeability-enhancing effects of VEGF and, thus, a major factor intumor angiogenesis; as a consequence, VEGFR2 overexpression can beobserved on tumor endothelial cells of angiogenic vessels in manycancers (Tucker G C (2006), Curr Oncol Rep 8, pp. 96-103; Parker et al.(2005), Protein Eng Des Sel 18, pp. 435-44; Boesen et al. (2002), J BiolChem 277, pp. 40335-41; Siemeister et al. (1998), Proc Natl Acad Sci USA95, pp. 4625-9; Cai et al. (2005), Biotechniques 39, pp. S6-S17; HaubnerR (2006), Eur J Nucl Med Mol Imaging 33 Suppl 1, pp. 54-63).

The term “scVEGF” as used herein describes a single-chain variant ofVEGF, particularly a single chain in which two “poles” of VEGF arejoined by a linker. For the purposes of the present invention the scVEGFis usually an antagonistic variant, as known in the art. Of particularrelevance is the study by Boesen, et al., supra., in which asingle-chain variant of VEGF121 (a common isoform of VEGF-A that doesnot require heparin binding like the larger isoform VEGF165 is preparedby linking the C-terminus of chain 1 to the N-terminus of chain 2 by a14-amino acid flexible linker. In addition, mutations are added to bothchains at one pole of the ligand in order to prevent binding of VEGFR2at one receptor-binding site. The result is a protein that can bind onlya single molecule of VEGFR2, and is antagonistic because it preventsreceptor dimerization and activation.

The VEGF dimer contains two receptor binding interfaces lying on eachpole of the molecule. Each of the two binding interfaces is typicallyable to contact one receptor monomer (either VEGFR1 or VEGFR2), therebyinducing receptor dimerization and activation. Consequently, anasymmetric VEGF variant that contains only one receptor bindinginterface at one pole of the dimer should not be able to induce receptordimerization and activation and, therefore, act as a VEGF antagonist(Siemeister et al. (1998), Proc Natl Acad Sci USA 95, pp. 4625-4629).

In certain embodiments the polypeptide of the invention is abifunctional single-chain antagonistic human VEGF variant comprising anative VEGF sequence, an amino acid linker, and a modified VEGF, wherethe modified VEGF comprises a loop with an inserted motif that binds toa vascular protein, which protein may include integrins such as αvβ3integrin, αvβ5, α5β1, etc., but may also include other vascular targets,e.g. including prostate membrane specific antigen (PMSA), PSA, MMPs,PDGFR, PDGF, and the like. Such polypeptides include without limitationany of the scVEGF polypeptides set forth herein, which further comprisethe modification of replacing amino acid residues of loop 2 or loop 3 inthe mutated VEGF pole with candidate motif. The binding motif may be apeptide sequence known in the art or may be designed through directedevolution, where a random or semi-random assortment of sequences isinserted into a permissive loop and screened for binding. In someembodiments the loop 3 sequence (SEQ ID NO:76) IKPHQGQ is replaced withthe motif. Binding of the modified polypeptide to a target may bedetermined by various methods, including selective binding to purifiedprotein, cell lines, tissues including sections of tumor tissue, and thelike.

Specific targets and motifs of interest include prostate specificmembrane antigen, which is a transmembrane glycoprotein homodimerexpressed almost exclusively in prostatic epithelial cells (O'Keefe D S,Prostate, 2004). Both expression and enzymatic activity of PSMA areelevated in prostate cancer and in the neovasculature of many solidtumors, with expression levels closely correlated with disease grade(Lapidus R G, Prostate, 2000). Interestingly, endothelial cells of theneovasculature of almost all solid tumors express PSMA but not cells inthe neovasculature associated with normal tissues (Silver D A, ClinCancer Res 1997). In particular, there is an increase in both expressionand enzymatic activity of PSMA in aggressive prostate tumors. Thehighest levels of PSMA expression are associated with high-grade,hormone-refractory and metastatic prostate cancer (Kawakami M, CancerRes., 1997). In fact, PSMA mRNA is upregulated upon androgen withdrawal(Israeli R S, Cancer Res., 1994). In general, PSMA expression isubiquitous, with expression in nearly all tumor sites. These propertieshave made PSMA an ideal target for developmental prostate cancer imagingagents and therapeutics, especially in advanced disease.

PSMA has both glutamate carboxypeptidase II activity that cleavesα-linked glutamate from N-acetylaspartyl glutamate (NAALADase activity)and γ-linked glutamates from polyglutamated folates sequentially (folatehydrolase activity). Although its mechanism in not yet known, PSMA (afolate hydrolase) may facilitate prostate carcinogenesis by enhancingthe proliferative and invasive capability of prostate cancer cells(which can be blocked by folic acid). It will be interesting to usepeptides that bind PSMA and can/cannot inhibit its enzymatic activity inorder to investigate if enzymatic activity contributes to the initiationof prostate carcinogenesis. It will also be interesting to see whetherthe active peptides will be able to block PSMA dimerization, which isdependent upon the presence of zinc ions in the active site of PSMA andis required for PSMA's enzymatic activity. This can be done using apurified ecto domain of PSMA which is able to dimerize (Lupoid S E, MolCancer Ther, 2004).

A stringent phage display strategy with a fusion protein containing onlythe extracellular portion of PSMA (containing two amino-terminalaffinity tags), was applied to identify potential PSMA binding peptides.Alignment revealed some weakly similar peptide sequences, providing theconsensus SEQ ID NO:76 VPHTR (Lupoid SE, Mol Cancer Ther, 2004). Themost active peptide SEQ ID NO:77 (CQIKHHNYLC) was able to bind purifiedPSMA (10 μM range), stabilize the protein to enhance enzymatic activity,and target phage to prostate cancer cells (LNCaP).

Linear peptides: In another study, a random phage library produced alinear peptide dimer SEQ ID NO:78 (WQPDTAHHWALT) with selectiveaffinities to prostate cancer cells expressing PSMA (LNCaP and CWR22R)vs. PSMA deficient cells. The peptide also had selective affinity topurified PSMA and ability to inhibit PSMA enzymatic activity (also inthe μM range) (Aggarwal S, Cancer Res., 2006). This dihistidine peptidemotif had also emerged as part of a consensus PSMA-binding sequence(i.e., SEQ ID NO:77 CQKHHNYLC) as mentioned above (Lupoid SE, Mol CancerTher, 2004). An interesting question to address will be whether thepresence of histidines, which are known to chelate divalent metal ionsincluding zinc (found in the PSMA catalytic binding site), may lead toinactivation of the enzyme and whether it depends on the sequence thatsurrounds them, which results in a specific fold that they adopt.

Other targets of interest include Matrix Metalloproteinases (MMPs).Tumor growth, angiogenesis, and metastasis are dependent on MMPactivity. However, the lack of inhibitors specific for the type IVcollagenase/gelatinase family of MMPs has thus far prevented theselective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) fortherapeutic intervention in cancer. Koivunan et al. (Koivunen E, NatureBiotechnology, 1999) used libraries of random peptides to isolateselective gelatinase inhibitors. They identified a class of cyclicpeptides containing an HWGF motif that are specific inhibitors of MMP-2and MMP-9.

Specifically, the cyclic decapeptide SEQ ID NO:79 CTTHWGFTLC was able to(i) inhibit the activities of these enzymes, (ii) suppress migration ofboth tumor cells and endothelial cells in vitro, (iii) home to tumorvasculature in vivo, and (iv) prevent the growth and invasion of tumorsin mice. SEQ ID NO:79 CTTHWGFTLC-displaying phage was also able tospecifically target angiogenic blood vessels in vivo.

“Integrins” are a family of cell surface adhesion receptors thatnon-covalently associate into α/β heterodimers with distinct ligandbinding specificities and cell signaling properties (Giancotti &Ruoslahti (1999), Science 285, pp. 1028-32). As cell surface adhesionreceptors, integrins are involved in the attachment of cells to matrixvia RGD peptide sequences; in addition, they function as receptors fortransmitting signals important for cell migration, invasion,proliferation, and survival. In their roles as major adhesion receptors,integrins signal across the plasma membrane in both directions. At leastsix integrin inhibitors on endothelial cells are being evaluated inclinical trials for cancer (Tucker (2006), Curr Oncol Rep 8, pp. 96-103)with αvβ3 (also known as the vitronectin receptor) being the mostabundant and influential receptor regulating angiogenesis (Shattil &Ginsberg (1997), J Clin Invest 100, pp. S91-S95).

There are several manifestations of a tightly collaborative relationshipbetween integrins and receptors for growth factors (Ross (2004),Cardiovasc Res 63, pp. 381-390). On endothelial cells, engagement ofαvβ3 integrin promotes phosphorylation and activation of vascularendothelial growth factor (VEGF) receptor (VEGFR)-2, thereby augmentingthe mitogenic activity of VEGFs (Soldi et al. (1999), EMBO J 18, pp.882-892). While αvβ3 integrins are highly expressed on activatedendothelial cells in tumor neovasculature, they are only weaklyexpressed in resting endothelial cells and most normal tissues andorgans (Brooks et al. (1994), Science 264, pp. 569-71; Brooks et al.(1994), Cell 79, pp. 1157-64). The terms “avb3”, “alpha v beta 3” and“αvβ3” are used interchangeably throughout the text.

RGD Peptides. It has been demonstrated that the αvβ3 integrin binds to anumber of Arg-Gly-Asp (RGD) containing matrix molecules, such asfibrinogen (Bennett et al. (1983), Proc Natl Acad Sci USA 80, p. 2417),fibronectin (Ginsberg et al. (1983), J Clin Invest 71, pp. 619-624), andvon Willebrand factor (Ruggeri et al. (1982), Proc Natl Acad Sci USA 79,p. 6038). Compounds containing the RGD sequence mimic extracellularmatrix ligands so as to bind to cell surface receptors.

While it has been fairly straightforward to insert RGD motifs intolinear or cyclic peptide libraries and screen for integrin binders withmicromolar affinities, generation of peptides that bind withtherapeutically relevant concentrations (low nanomolar) or highspecificities to particular integrins require that the RGD sequence isappropriately positioned for binding the integrin of interest. Likenatural integrin ligands, the affinities and specificities of theseRGD-containing peptides and proteins are largely dependent on theorientation of the Arg and Asp residues, as well as the conformation ofthe RGD loop, which is dictated by the amino acids flanking the RGDsequence. Rigidifying the RGD motif by backbone cyclization or placingit within a disulfide-bonded loop can improve integrin-binding affinityand specificity (Silverman et al. (2009), J Mol Biol 385, pp. 1064-1075.

The term “domain” as used herein describes a discrete portion of aprotein assumed to fold independently of the rest of the protein andpossessing its own function. The term “single domain” as used hereindescribes the presence of one domain in a protein.

The terms “polypeptide” and “polypeptides” as used herein includeproteins and fragments thereof. Polypeptides are disclosed herein asamino acid residue sequences. Those sequences are written left to rightin the direction from the amino to the carboxy terminus or N to Cterminus. In accordance with standard nomenclature, amino acid residuesequences are denominated by either a three letter or a single lettercode as indicated as follows: Alanine (Ala, A), Arginine (Arg, R),Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C),Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine(His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K),Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine(Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y),and Valine (Val, V).

The term “variant” refers to a polypeptide or protein that differs froma reference polypeptide or protein, but retains essential properties. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall (homologous) and, in many regions, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more modifications (e.g., substitutions, additions, and/ordeletions). A substituted or inserted amino acid residue may or may notbe one encoded by the genetic code. A variant of a polypeptide may benaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. The term “identical or essentiallysimilar single-chain VEGF variants” as used herein include variantshaving more than 50% sequence identity to the single-chain VEGF variantsdisclosed in embodiments of the present invention.

The terms “mutant” and “clone” are employed broadly to refer to aprotein that differs in some way from a reference wild-type protein,where the protein may retain biological properties of the referencewild-type (e.g., naturally occurring) protein, or may have biologicalproperties that differ from the reference wild-type protein. For thepurposes of the invention reference may be made to a “modified VEGFreceptor binding site”, which differs in amino acid sequence from thenative polypeptide but which retains properties of interest. The term“biological property” of the subject proteins includes, but is notlimited to, biological interactions in cancer and/or ischemic or hypoxicrelated diseases, in vivo and/or in vitro stability (e.g., half-life),and the like. Mutants and clones can include single amino acid changes(point mutations), deletions of one or more amino acids(point-deletions), N-terminal truncations, C-terminal truncations,insertions, and the like. Mutants and clones can be generated usingstandard techniques of molecular biology.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are herein described.

Modifications and changes can be made in the structure of thepolypeptides and proteins of this disclosure and still result in amolecule having similar characteristics as the polypeptide (e.g., aconservative amino acid substitution). For example, certain amino acidscan be substituted for other amino acids in a sequence withoutappreciable loss of activity. Because it is the interactive capacity andnature of a polypeptide that defines that polypeptide's or protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a polypeptide or protein sequence andnevertheless obtain a polypeptide or protein with like properties.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take one or more of the foregoing characteristicsinto consideration are well known to those of skill in the art andinclude, but are not limited to (original residue: exemplarysubstitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu,Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile:Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr),(Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu).Embodiments of this disclosure, therefore, consider functional orbiological equivalents of a polypeptide or protein as set forth above.In particular, embodiments of the polypeptides and proteins can includevariants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identityto the polypeptide and protein of interest.

“Identity,” as known in the art, is a relationship between two or morepolypeptide or protein sequences, as determined by comparing thesequences. In the art, “identity” also refers to the degree of sequencerelatedness between polypeptides or proteins, as determined by the matchbetween strings of such sequences. “Identity” can be readily calculatedby known bioinformational methods.

Polypeptide Compositions

Provided herein are compositions and methods related to single-chainvariants of VEGF, including bifunctional proteins targeting both VEGFR2and αvβ3 integrin, effectively antagonizing their activation and soexerting anti-angiogenic effects. As single-chain antagonistic VEGFvariants with one intact VEGF receptor binding site at the one pole andone mutated VEGF receptor binding site at the other pole, these proteinsbind to VEGF receptors, in particular to VEGFR2 receptors, but fail toinduce receptor activation, thereby antagonizing VEGF-stimulatedreceptor autophosphorylation and proliferation of endothelial cells. Inaddition, single-chain antagonistic VEGF variants may comprise a loopcarrying an integrin-recognition RGD sequence for binding of αvβ3integrin in the mutated receptor binding site, thereby antagonizing notonly VEGF-stimulated receptor autophosphorylation and proliferation ofendothelial cells, but also the activation of alpha v beta 3 integrin.

Since VEGFR1 and VEGFR2 belong to the class of oligomeric cellularreceptors that depend on oligomerization and/or conformational changesto be activated, binding of the single-chain variants of the presentinvention without activation of the receptors allows the single-chainvariants to function as effective antagonists of VEGF and VEGF-mediatedphosphorylation and stimulation of endothelial cells.

Embodiments of the invention describe the preparation of suchbifunctional, single-chain VEGF variants and their use in molecularcancer imaging and treatment of cancer, age-related maculardegeneration, diabetic retinopathy, rheumatoid arthritis and psoriasis.

Single-chain antagonistic VEGF protein variants were engineered from themonomer VEGF-121, but contain only the 97-amino acid core region ofVEGF-121, and have truncated N- and C-termini relative to VEGF-121.These variants have one intact and one mutated VEGF receptor bindingsites, where the mutated binding site contains a loop with anintegrin-recognition RGD sequence for binding of alpha v beta 3integrin. The single-chain VEGF variants bind to VEGF receptors, inparticular to VEGFR2 receptors, but fail to induce receptor activation,thereby antagonizing VEGF-stimulated receptor autophosphorylation andproliferation of endothelial cells.

An exemplary single chain variant of VEGF comprises two chains of the97-amino acid core region of VEGF-121 (E13-D109). A flexible, amino acidlinker links the C-terminus of chain 1 to the N-terminus of chain 2. Anexemplary linker comprises the amino acid sequence SEQ ID NO:80GSTSGSGKSSEGKG, however many such linkers are known and used in the artand may serve this purpose. The polypeptides of the invention aretypically provided in single-chain form, which means that the monomersare linked by peptide bonds through a linker peptide, rather than beinglinked by noncovalent bonds or disulfide bonds. Optionally Chain 1 hasF17A and E64A mutations in the VEGFR2 recognition region. Chain 2may bemutated to abolish binding to VEGFR2, including without limitation anI46A mutation, I83A mutation, etc.

In some embodiments the polypeptide of the invention is a single-chainantagonistic human VEGF variant having increased affinity for theVEGF2R, relative to the native polypeptide. Such polypeptides includewithout limitation those set forth in SEQ ID NO:9-18.

In some embodiments the polypeptide of the invention is a bifunctionalsingle-chain antagonistic human VEGF variant comprising a native VEGFsequence, an amino acid linker, and a modified VEGF, where the modifiedVEGF comprises a loop with an integrin-recognition RGD sequence capableof binding αvβ3 integrin. Such polypeptides include without limitationthose set forth in SEQ ID NO:5-8. Such polypeptides also include anypolypeptide of SEQ ID NO:9-18 and 19-27, further comprising themodification of replacing amino acid residues of loop 3 in the mutatedVEGF pole with an RGD motif, which RGD motif includes, withoutlimitation XXRGDXXXX, XXXRGDXXX, or XXXXRGDXX, where X is any aminoacid. Specific RGD motifs of interest include those set forth in SEQ IDNO:29-SEQ ID NO:75. In some embodiments the loop 3 sequence (SEQ IDNO:76) IKPHQGQ (I83-Q89) is replaced with the RGD motif.

In some embodiments the polypeptide of the invention is a bifunctionalsingle-chain antagonistic human VEGF variant having increased affinityfor the VEGFR2, relative to the native polypeptide. Such polypeptidesinclude without limitation those set forth in SEQ ID NO:19-27.

Table 1 shows all of the sequences of the integrin-binding loop peptidesutilized in the polypeptides of the invention.

Grafted Loop Sequence SEQ ID NO: 29 PFGTRGDSS SEQ ID NO: 30 SGERGDGPTSEQ ID NO: 31 SDGRGDGSV SEQ ID NO: 32 PIGRGDGST SEQ ID NO: 33 LAERGDSSSSEQ ID NO: 34 PTGRGDLGA SEQ ID NO: 35 RGIRGDSGA SEQ ID NO: 36 VGGRGDVGVSEQ ID NO: 37 ITARGDSFG SEQ ID NO: 38 ITERGDSGH SEQ ID NO: 39 PQARGDRSDSEQ ID NO: 40 SRTRGDASD SEQ ID NO: 41 PAARGDGGL SEQ ID NO: 42 PVARGDSGASEQ ID NO: 43 PQQRGDGPH SEQ ID NO: 44 PLPRGDGQR SEQ ID NO: 45 HAGRGDSPSSEQ ID NO: 46 TSLRGDTTW SEQ ID NO: 47 PNFRGDEAY SEQ ID NO: 48 AGVPRGDSPSEQ ID NO: 49 PRSTRGDST SEQ ID NO: 50 PFGVRGDDN SEQ ID NO: 51GFPFRGDSPAS SEQ ID NO: 52 PSVRRGDSPAS SEQ ID NO: 53 PFAVRGDRPSEQ ID NO: 54 PWPRRGDLP SEQ ID NO: 55 PSGGRGDSP SEQ ID NO: 56 VGGRGDVGVSEQ ID NO: 57 ITSRGDHGE SEQ ID NO: 58 PPGRGDNGG SEQ ID NO: 59 PVARGDSGASEQ ID NO: 60 STDRGDASA SEQ ID NO: 61 LNPRGDANT SEQ ID NO: 62PSVRRGDSPAS SEQ ID NO: 63 PTTRGDCPD SEQ ID NO: 64 PGGRGDSAYSEQ ID NO: 65 PHDRGDAGV SEQ ID NO: 66 STDRGDASA SEQ ID NO: 67 ASGRGDGGVSEQ ID NO: 68 PASRGDSPP

Modifications and changes can be made in the selection of the monomersused (VEGF-121, VEGF-145, VEGF-165, VEGF-189 and VEGF-206), in thelength of the core region of VEGF and/or in the length of the linkeryielding identical or essentially similar single-chain VEGF variantswith like properties as for the single-chain VEGF variants described inembodiments of the present invention.

Polypeptides can be produced through recombinant methods and chemicalsynthesis. In addition, functionally equivalent polypeptides may finduse, where the equivalent polypeptide may contain deletions, additionsor substitutions of amino acid residues that result in a silent change,thus producing a functionally equivalent differentially expressed onpathway gene product. Amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.“Functionally equivalent”, as used herein, refers to a protein capableof exhibiting a substantially similar in vivo activity.

The polypeptides may be produced by recombinant DNA technology usingtechniques well known in the art. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. Alternatively, RNAcapable of encoding the polypeptides of interest may be chemicallysynthesized.

Typically, the coding sequence is placed under the control of a promoterthat is functional in the desired host cell to produce relatively largequantities of the gene product. An extremely wide variety of promotersare well-known, and can be used in the expression vectors of theinvention, depending on the particular application. Ordinarily, thepromoter selected depends upon the cell in which the promoter is to beactive. Other expression control sequences such as ribosome bindingsites, transcription termination sites and the like are also optionallyincluded. Constructs that include one or more of these control sequencesare termed “expression cassettes.” Expression can be achieved inprokaryotic and eukaryotic cells utilizing promoters and otherregulatory agents appropriate for the particular host cell. Exemplaryhost cells include, but are not limited to, E. coli, other bacterialhosts, yeast, and various higher eukaryotic cells such as the COS, CHOand HeLa cells lines and myeloma cell lines.

The polypeptide may be labeled, either directly or indirectly. Any of avariety of suitable labeling systems may be used, including but notlimited to, radioisotopes such as ¹²⁵I; enzyme labeling systems thatgenerate a detectable colorimetric signal or light when exposed tosubstrate; and fluorescent labels. Indirect labeling involves the use ofa protein, such as a labeled antibody, that specifically binds to thepolypeptide of interest. Such antibodies include but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression library.

Once expressed, the recombinant polypeptides can be purified accordingto standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, ion exchange and/or size exclusivitychromatography, gel electrophoresis and the like (see, generally, R.Scopes, Protein Purification, Springer—Verlag, N.Y. (1982), Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification., AcademicPress, Inc. N.Y. (1990)).

As an option to recombinant methods, polypeptides can be chemicallysynthesized. Such methods typically include solid-state approaches, butcan also utilize solution based chemistries and combinations orcombinations of solid-state and solution approaches. Examples ofsolid-state methodologies for synthesizing proteins are described byMerrifield (1964) J. Am. Chem. Soc. 85:2149; and Houghton (1985) Proc.Natl. Acad. Sci., 82:5132. Fragments of polypeptides of the inventionprotein can be synthesized and then joined together. Methods forconducting such reactions are described by Grant (1992) SyntheticPeptides: A User Guide, W.H. Freeman and Co., N.Y.; and in “Principlesof Peptide Synthesis,” (Bodansky and Trost, ed.), Springer-Verlag, Inc.N.Y., (1993).

The polypeptides of the invention can be coupled or conjugated to one ormore cytotoxic or imaging moieties. As used herein, “cytotoxic moiety”is a moiety that inhibits cell growth or promotes cell death whenproximate to or absorbed by the cell. Suitable cytotoxic moieties inthis regard include radioactive isotopes (radionuclides), chemotoxicagents such as differentiation inducers and small chemotoxic drugs,toxin proteins, and derivatives thereof. “Imaging moiety” (I) is amoiety that can be utilized to increase contrast between a tumor and thesurrounding healthy tissue in a visualization technique (e.g.,radiography, positron-emission tomography, single-photon emissioncomputed tomography, near-infrared fluorescence imaging, magneticresonance imaging, ultrasound, direct or indirect visual inspection).Thus, suitable imaging moieties include radiography moieties (e.g. heavymetals and radiation emitting moieties), positron emitting moieties,magnetic resonance contrast moieties, gas-filled microbubble spheres forcontrast-enhanced ultrasound, and optically visible moieties (e.g.,fluorescent or visible-spectrum dyes, visible particles, etc.). It willbe appreciated by one of ordinary skill that some overlap exists betweentherapeutic and imaging moieties. For instance ²¹²Pb and ²¹²Bi are bothuseful radioisotopes for therapeutic compositions, but are alsoelectron-dense, and thus provide contrast for X-ray radiographic imagingtechniques, and can also be utilized in scintillation imagingtechniques.

In general, therapeutic or imaging agents may be conjugated to thepolypeptides of the invention by any suitable technique, withappropriate consideration of the need for pharmokinetic stability andreduced overall toxicity to the patient. A therapeutic agent may becoupled to a polypeptide either directly or indirectly (e.g. via alinker group). A direct reaction between an agent and a polypeptide ispossible when each possesses a functional group capable of reacting withthe other. For example, a nucleophilic group, such as an amino orsulfhydryl group, may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide). Alternatively, asuitable chemical linker group may be used. A linker group can functionas a spacer to distance a polypeptide from an agent in order to avoidinterference with binding capabilities. A linker group can also serve toincrease the chemical reactivity of a substituent on a moiety or apolypeptide, and thus increase the coupling efficiency. An increase inchemical reactivity may also facilitate the use of moieties, orfunctional groups on moieties, which otherwise would not be possible.

Suitable linkage chemistries include maleimidyl linkers and alkyl halidelinkers (which react with a sulfhydryl on the polypeptide moiety) andsuccinimidyl linkers (which react with a primary amine on thepolypeptide moiety). Several primary amine and sulfhydryl groups arepresent on a polypeptide, and additional groups may be designed intorecombinant molecules. It will be evident to those skilled in the artthat a variety of bifunctional or polyfunctional reagents, both homo-and hetero-functional (such as those described in the catalog of thePierce Chemical Co., Rockford, Ill.), may be employed as a linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958. As an alternative coupling method, cytotoxic or imagingmoieties may be coupled to the polypeptides of the invention through anoxidized carbohydrate group at a glycosylation site, as described inU.S. Pat. Nos. 5,057,313 and 5,156,840. Yet another alternative methodof coupling a polypeptide to the cytotoxic or imaging moiety is by theuse of a non-covalent binding pair, such as streptavidin/biotin, oravidin/biotin. In these embodiments, one member of the pair iscovalently coupled to a polypeptide and the other member of the bindingpair is covalently coupled to the cytotoxic or imaging moiety.

Carriers and linkers specific for radionuclide agents (both for use ascytotoxic moieties or positron-emission imaging moieties) includeradiohalogenated small molecules and chelating compounds. For example,U.S. Pat. No. 4,735,792 discloses representative radiohalogenated smallmolecules and their synthesis. A radionuclide chelate may be formed fromchelating compounds that include those containing nitrogen and sulfuratoms as the donor atoms for binding the metal, or metal oxide,radionuclide. Such chelation carriers are also useful for magnetic spincontrast ions for use in magnetic resonance imaging tumor visualizationmethods, and for the chelation of heavy metal ions for use inradiographic visualization methods.

Preferred radionuclides for use as cytotoxic moieties are radionuclidesthat are suitable for pharmacological administration. Such radionuclidesinclude ¹²³I, ¹²⁵I, ¹³¹I, ⁹⁰Y, ²¹¹At, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, and²¹²Bi. Iodine and astatine isotopes are more preferred radionuclides foruse in the therapeutic compositions of the present invention, as a largebody of literature has been accumulated regarding their use. ¹³¹I isparticularly preferred, as are other β-radiation emitting nuclides,which have an effective range of several millimeters. ¹²³I, ¹²⁵I, ¹³¹I,or ²¹¹At may be conjugated to polypeptides of the invention for use inthe compositions and methods utilizing any of several known conjugationreagents, including Iodogen, N-succinimidyl 3-[²¹¹At]astatobenzoate,N-succinimidyl 3-[¹³¹I]iodobenzoate (SIB), and N-succinimidyl5-[¹³¹I]iodob-3-pyridinecarboxylate (SIPC). Any iodine isotope may beutilized in the recited iodo-reagents. Radionuclides can be conjugatedto polypeptides of the invention by suitable chelation agents known tothose of skill in the nuclear medicine arts.

Preferred radiographic moieties for use as imaging moieties in thepresent invention include compounds and chelates with relatively largeatoms, such as gold, iridium, technetium, barium, thallium, iodine, andtheir isotopes. It is preferred that less toxic radiographic imagingmoieties, such as iodine or iodine isotopes, be utilized in thecompositions and methods of the invention. Examples of suchcompositions, which may be utilized for x-ray radiography are describedin U.S. Pat. No. 5,709,846, incorporated fully herein by reference. Suchmoieties may be conjugated to the polypeptides of the invention throughan acceptable chemical linker or chelation carrier. In addition,radionuclides which emit radiation capable of penetrating the skull maybe useful for scintillation imaging techniques. Suitable radionuclidesfor conjugation include ⁹⁹Tc, ¹¹¹In, and ⁶⁷Ga. Positron emittingmoieties for use in the present invention include ¹⁸F, which can beeasily conjugated by a fluorination reaction with the polypeptides ofthe invention according to the method described in U.S. Pat. No.6,187,284, or ⁶⁴Cu, which can be conjugated through chemical chelatorsas extensively described in the literature.

Preferred magnetic resonance contrast moieties include chelates ofchromium(III), manganese(II), iron(II), nickel(II), copper(II),praseodymium(III), neodymium(III), samarium(III) and ytterbium(III) ion.Because of their very strong magnetic moment, the gadolinium(III),terbium(III), dysprosium(III), holmium(III), erbium(III), and iron(III)ions are especially preferred. Examples of such chelates, suitable formagnetic resonance spin imaging, are described in U.S. Pat. No.5,733,522, incorporated fully herein by reference. Nuclear spin contrastchelates may be conjugated to the polypeptides of the invention througha suitable chemical linker.

Optically visible moieties for use as imaging moieties includefluorescent dyes, or visible-spectrum dyes, visible particles, and othervisible labeling moieties. Fluorescent dyes such as ALEXA dyes,fluorescein, coumarin, rhodamine, bodipy Texas red, and cyanine dyes,are useful when sufficient excitation energy can be provided to the siteto be inspected visually. Endoscopic visualization procedures may bemore compatible with the use of such labels. For many procedures whereimaging agents are useful, such as during an operation to resect a braintumor, visible spectrum dyes are preferred. Acceptable dyes includeFDA-approved food dyes and colors, which are non-toxic, althoughpharmaceutically acceptable dyes which have been approved for internaladministration are preferred. In preferred embodiments, such dyes areencapsulated in carrier moieties, which are in turn conjugated to thepolypeptides of the invention. Alternatively, visible particles, such ascolloidal gold particles or latex particles, may be coupled to thepolypeptides of the invention via a suitable chemical linker.

Pharmaceutical Formulations

Formulations of polypeptides of the invention find use in diagnosis andtherapy. The formulation may comprise one, two or more polypeptides ofthe invention. The therapeutic formulation may be administered incombination with other methods of treatment, e.g. chemotherapy,radiation therapy, surgery, and the like.

Formulations may be optimized for retention and stabilization at atargeted site. Stabilization techniques include enhancing the size ofthe polypeptide, by cross-linking, multimerizing, or linking to groupssuch as polyethylene glycol, polyacrylamide, neutral protein carriers,etc. in order to achieve an increase in molecular weight. Otherstrategies for increasing retention include the entrapment of thepolypeptide in a biodegradable or bioerodible implant. The rate ofrelease of the therapeutically active agent is controlled by the rate oftransport through the polymeric matrix, and the biodegradation of theimplant. The transport of polypeptide through the polymer barrier willalso be affected by compound solubility, polymer hydrophilicity, extentof polymer cross-linking, expansion of the polymer upon water absorptionso as to make the polymer barrier more permeable to the drug, geometryof the implant, and the like. The implants are of dimensionscommensurate with the size and shape of the region selected as the siteof implantation. Implants may be particles, sheets, patches, plaques,fibers, microcapsules and the like and may be of any size or shapecompatible with the selected site of insertion.

Pharmaceutical compositions can include, depending on the formulationdesired, pharmaceutically-acceptable, non-toxic carriers of diluents,which are defined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, buffered water, physiologicalsaline, PBS, Ringer's solution, dextrose solution, and Hank's solution.In addition, the pharmaceutical composition or formulation can includeother carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenicstabilizers, excipients and the like. The compositions can also includeadditional substances to approximate physiological conditions, such aspH adjusting and buffering agents, toxicity adjusting agents, wettingagents and detergents.

The composition can also include any of a variety of stabilizing agents,such as an antioxidant for example. The polypeptide may be complexedwith various well-known compounds that enhance the in vivo stability ofthe polypeptide, or otherwise enhance its pharmacological properties(e.g., increase the half-life of the polypeptide, reduce its toxicity,enhance solubility or uptake). Examples of such modifications orcomplexing agents include sulfate, gluconate, citrate and phosphate. Thepolypeptides of a composition can also be complexed with molecules thatenhance their in vivo attributes. Such molecules include, for example,carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,sodium, potassium, calcium, magnesium, manganese), and lipids.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

The pharmaceutical compositions can be administered for prophylacticand/or therapeutic treatments. Toxicity and therapeutic efficacy of theactive ingredient can be determined according to standard pharmaceuticalprocedures in cell cultures and/or experimental animals, including, forexample, determining the LD₅₀ (the dose lethal to 50% of the population)and the ED₅₀ (the dose therapeutically effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred.

The data obtained from cell culture and/or animal studies can be used informulating a range of dosages for humans. The dosage of the activeingredient typically lines within a range of circulating concentrationsthat include the ED₅₀ with low toxicity. The dosage can vary within thisrange depending upon the dosage form employed and the route ofadministration utilized.

The pharmaceutical compositions described herein can be administered ina variety of different ways. Examples include administering acomposition containing a pharmaceutically acceptable carrier via oral,intranasal, rectal, topical, intraperitoneal, intravenous,intramuscular, subcutaneous, subdermal, transdermal, intrathecal, andintracranial methods.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, intraperitoneal,and subcutaneous routes, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Nucleic Acids

Nucleic acid sequences encoding polypeptides of the invention find usein the recombinant production of the encoded polypeptide, and the like.One of skill in the art can readily utilize well-known codon usagetables and synthetic methods to provide a suitable coding sequence forany of the polypeptides of the invention. Direct chemical synthesismethods include, for example, the phosphotriester method of Narang etal. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brownet al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramiditemethod of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and thesolid support method of U.S. Pat. No. 4,458,066. Chemical synthesisproduces a single stranded oligonucleotide. This can be converted intodouble stranded DNA by hybridization with a complementary sequence, orby polymerization with a DNA polymerase using the single strand as atemplate. While chemical synthesis of DNA is often limited to sequencesof about 100 bases, longer sequences can be obtained by the ligation ofshorter sequences. Alternatively, subsequences may be cloned and theappropriate subsequences cleaved using appropriate restriction enzymes.

The nucleic acids of the subject invention are isolated and obtained insubstantial purity, generally as other than an intact chromosome.Usually, the nucleic acids, either as DNA or RNA, will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant,” e.g., flanked by one or more nucleotideswith which it is not normally associated on a naturally occurringchromosome. The nucleic acids of the invention can be provided as alinear molecule or within a circular molecule, and can be providedwithin autonomously replicating molecules (vectors) or within moleculeswithout replication sequences. Expression of the nucleic acids can beregulated by their own or by other regulatory sequences known in theart. The nucleic acids of the invention can be introduced into suitablehost cells using a variety of techniques available in the art, such astransferrin polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

Methods of Use

Molecular imaging unites molecular biology and in vivo imaging. Itenables the visualisation of the cellular function and the follow-up ofthe molecular process in living organisms without perturbing them.

In some embodiments, the methods are adapted for imaging use in vivo,e.g., to locate or identify sites where angiogenic cells are present. Inthese embodiments, a detectably-labeled polypeptide of the invention isadministered to an individual (e.g., by injection), and labeled cellsare located using standard imaging techniques, including, but notlimited to, near-infrared fluorescence imaging, positron emissiontomography, magnetic resonance imaging, computed tomography scanning,and the like.

For diagnostic in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given radionuclide. Theradionuclide chosen must have a type of decay that is detectable by agiven type of instrument. In general, any conventional method forvisualizing diagnostic imaging can be utilized in accordance with thisinvention. Another important factor in selecting a radionuclide for invivo diagnosis is that its half-life be long enough that it is stilldetectable at the time of maximum uptake by the target tissue, but shortenough that deleterious radiation of the host is minimized. A currentlyused method for labeling with ^(99m)Tc is the reduction of pertechnetateion in the presence of a chelating precursor to form the labile^(99m)Tc-precursor complex, which, in turn, reacts with the metalbinding group of a bifunctionally modified chemotactic peptide to form a^(99m)Tc-chemotactic peptide conjugate. In one embodiment, the imagingmethod is one of PET or SPECT, which are imaging techniques in which aradionuclide is synthetically or locally administered to a patient. Thesubsequent uptake of the radiotracer is measured over time and used toobtain information about the targeted tissue. Because of the high-energy(γ-ray) emissions of the specific isotopes employed and the sensitivityand sophistication of the instruments used to detect them, thetwo-dimensional distribution of radioactivity may be inferred fromoutside of the body. Among the most commonly used positron-emittingnuclides in PET are included ¹¹C, ¹³N, ¹⁵O, and ¹⁸F, and ⁶⁴Cu. Isotopesthat decay by electron capture and/or γ emission are used in SPECT, andinclude ¹²³I and ^(99m)Tc, and ¹¹¹In.

Therapeutic Methods

The dose of a polypeptide of the invention administered to a subject,particularly a human, in the context of the present invention should besufficient to effect a therapeutic reduction in angiogenesis in thesubject over a reasonable time frame. The dose will be determined by,among other considerations, the potency of the particular polypeptide ofthe invention employed and the condition of the subject, as well as thebody weight of the subject to be treated. The size of the dose also willbe determined by the existence, nature, and extent of any adverseside-effects that might accompany the administration of a particularcompound.

In determining the effective amount of polypeptide in the reduction ofangiogenesis, the route of administration, the kinetics of the releasesystem (e.g., pill, gel or other matrix), and the potency of the agonistare considered so as to achieve the desired anti-angiogenic effect withminimal adverse side effects. The polypeptide of the invention willtypically be administered to the subject being treated for a time periodranging from a day to a few weeks, consistent with the clinicalcondition of the treated subject.

As will be readily apparent to the ordinarily skilled artisan, thedosage is adjusted for polypeptide of the invention according to theirpotency and/or efficacy relative to a VEGF antagonist. A dose may be inthe range of about 0.001 μg to 100 mg, given 1 to 20 times daily, andcan be up to a total daily dose of about 0.01 μg to 100 mg. If appliedtopically, for the purpose of a systemic effect, the patch or creamwould be designed to provide for systemic delivery of a dose in therange of about 0.01 μg to 100 mg. If injected for the purpose of asystemic effect, the matrix in which the polypeptide of the invention isadministered is designed to provide for a systemic delivery of a dose inthe range of about 0.001 μg to 1 mg. If injected for the purpose of alocal effect, the matrix is designed to release locally an amount ofpolypeptide of the invention in the range of about 0.001 μg to 100 mg.

Regardless of the route of administration, the dose of polypeptide ofthe invention can be administered over any appropriate time period,e.g., over the course of 1 to 24 hours, over one to several days, etc.Furthermore, multiple doses can be administered over a selected timeperiod. A suitable dose can be administered in suitable subdoses perday, particularly in a prophylactic regimen. The precise treatment levelwill be dependent upon the response of the subject being treated.

In some embodiments, a polypeptide of the invention is administered in acombination therapy with one or more other therapeutic agents, includingan inhibitor of angiogenesis; and a cancer chemotherapeutic agent.

Suitable chemotherapeutic agents include, but are not limited to, thealkylating agents, e.g. Cisplatin, Cyclophosphamide, Altretamine; theDNA strand-breakage agents, such as Bleomycin; DNA topoisomerase IIinhibitors, including intercalators, such as Amsacrine, Dactinomycin,Daunorubicin, Doxorubicin, Idarubicin, and Mitoxantrone; thenonintercalating topoisomerase II inhibitors such as, Etoposide andTeniposide; the DNA minor groove binder Plicamycin; alkylating agents,including nitrogen mustards such as Chlorambucil, Cyclophosphamide,Isofamide, Mechlorethamine, Melphalan, Uracil mustard; aziridines suchas Thiotepa; methanesulfonate esters such as Busulfan; nitroso ureas,such as Carmustine, Lomustine, Streptozocin; platinum complexes, such asCisplatin, Carboplatin; bioreductive alkylator, such as Mitomycin, andProcarbazine, Dacarbazine and Altretamine; antimetabolites, includingfolate antagonists such as Methotrexate and trimetrexate; pyrimidineantagonists, such as Fluorouracil, Fluorodeoxyuridine, CB3717,Azacytidine, Cytarabine; Floxuridine purine antagonists includingMercaptopurine, 6-Thioguanine, Fludarabine, Pentostatin; sugar modifiedanalogs include Cyctrabine, Fludarabine; ribonucleotide reductaseinhibitors including hydroxyurea; Tubulin interactive agents includingVincristine Vinblastine, and Paclitaxel; adrenal corticosteroids such asPrednisone, Dexamethasone, Methylprednisolone, and Prodnisolone;hormonal blocking agents including estrogens, conjugated estrogens andEthinyl Estradiol and Diethylstilbesterol, Chlorotrianisene andIdenestrol; progestins such as Hydroxyprogesterone caproate,Medroxyprogesterone, and Megestrol; androgens such as testosterone,testosterone propionate; fluoxymesterone, methyltestosterone estrogens,conjugated estrogens and Ethinyl Estradiol and Diethylstilbesterol,Chlorotrianisene and Idenestrol; progestins such as Hydroxyprogesteronecaproate, Medroxyprogesterone, and Megestrol; androgens such astestosterone, testosterone propionate; fluoxymesterone,methyltestosterone; and the like.

The polypeptide of the invention may be administered with otheranti-angiogenic agents. Anti-angiogenic agents include, but are notlimited to, angiostatic steroids such as heparin derivatives andglucocorticosteroids; thrombospondin; cytokines such as IL-12;fumagillin and synthetic derivatives thereof, such as AGM 12470;interferon-α; endostatin; soluble growth factor receptors; neutralizingmonoclonal antibodies directed against growth factors such as vascularendothelial growth factor; and the like.

The instant invention provides a method of reducing angiogenesis in amammal. The method generally involves administering to a mammal apolypeptide of the invention in an amount effective to reduceangiogenesis. An effective amount of an polypeptide of the inventionreduces angiogenesis by at least about 10%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,or more, when compared to an untreated (e.g., a placebo-treated)control.

Whether angiogenesis is reduced can be determined using any knownmethod. Methods of determining an effect of an agent on angiogenesis areknown in the art and include, but are not limited to, inhibition ofneovascularization into implants impregnated with an angiogenic factor;inhibition of blood vessel growth in the cornea or anterior eye chamber;inhibition of endothelial cell proliferation, migration or tubeformation in vitro; the chick chorioallantoic membrane assay; thehamster cheek pouch assay; the polyvinyl alcohol sponge disk assay. Suchassays are well known in the art and have been described in numerouspublications, including, e.g., Auerbach et al. ((1991) Pharmac. Ther.51:1-11), and references cited therein.

The invention further provides methods for treating a condition ordisorder associated with or resulting from pathological angiogenesis. Inthe context of cancer therapy, a reduction in angiogenesis according tothe methods of the invention effects a reduction in tumor size; and areduction in tumor metastasis. Whether a reduction in tumor size isachieved can be determined, e.g., by measuring the size of the tumor,using standard imaging techniques. Whether metastasis is reduced can bedetermined using any known method. Methods to assess the effect of anagent on tumor size are well known, and include imaging techniques suchas computerized tomography and magnetic resonance imaging.

Any condition or disorder that is associated with or that results frompathological angiogenesis, or that is facilitated by neovascularization(e.g., a tumor that is dependent upon neovascularization), is amenableto treatment with a polypeptide of the invention.

Conditions and disorders amenable to treatment include, but are notlimited to, cancer; atherosclerosis; proliferative retinopathies such asdiabetic retinopathy, age-related maculopathy, retrolental fibroplasia;excessive fibrovascular proliferation as seen with chronic arthritis;psoriasis; and vascular malformations such as hemangiomas, and the like.

The instant methods are useful in the treatment of both primary andmetastatic solid tumors, including carcinomas, sarcomas, leukemias, andlymphomas. Of particular interest is the treatment of tumors occurringat a site of angiogenesis. Thus, the methods are useful in the treatmentof any neoplasm, including, but not limited to, carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract,(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma) and tumors ofthe brain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas). The instant methods are also useful for treating solidtumors arising from hematopoietic malignancies such as leukemias (i.e.chloromas, plasmacytomas and the plaques and tumors of mycosis fungoidesand cutaneous T-cell lymphoma/leukemia) as well as in the treatment oflymphomas (both Hodgkin's and non-Hodgkin's lymphomas). In addition, theinstant methods are useful for reducing metastases from the tumorsdescribed above either when used alone or in combination withradiotherapy and/or other chemotherapeutic agents.

Other conditions and disorders amenable to treatment using the methodsof the instant invention include autoimmune diseases such as rheumatoid,immune and degenerative arthritis; various ocular diseases such asdiabetic retinopathy, retinopathy of prematurity, corneal graftrejection, retrolental fibroplasia, neovascular glaucoma, rubeosis,retinal neovascularization due to macular degeneration, hypoxia,angiogenesis in the eye associated with infection or surgicalintervention, and other abnormal neovascularization conditions of theeye; skin diseases such as psoriasis; blood vessel diseases such ashemangiomas, and capillary proliferation within atherosclerotic plaques;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; and excessive wound granulation(keloids).

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible. In thefollowing, examples will be described to illustrate parts of theinvention.

EXAMPLES Example 1 Generation of Single-chain VEGF Variant

We created a single-chain variant of VEGF (termed scVEGF) in which twomonomeric VEGF chains were physically tethered through a flexiblelinker. Point mutations were introduced into scVEGF (chain 1: F17A,E64A; chain 2: I46A, I83A) that confer antagonistic activity by blockinga second molecule of VEGFR1 or VEGFR2 from binding (Boesen et al.(2002), J Biol Chem 277, pp. 40335-41; Siemeister et al. (1998), ProcNatl Acad Sci USA 95, pp. 4625-9; Fuh et al. (1998), J Biol Chem 273,pp. 11197-204). Once single-chain VEGF variants were established, a 9amino-acid integrin binding loop was introduced into scVEGF in place ofresidues 183-089 (on chain 2), which in wild-type VEGF would normallyallow binding a second molecule of VEGFR2.

Example 2 Single-chain VEGF Antagonists Engineered to BispecificallyTarget VEGFR2 and α_(v)β₃ Integrin

In this work, we used a single-chain VEGF (scVEGF) antagonist as ascaffold for engineering recognition to integrin αvβ3. The mutated poleof scVEGF had one of the VEGFR2-recognition loops replaced with a loopcontaining RGD and randomized flanking residues. The library ofscVEGFrgd variants was screened by yeast surface display, and proteinswith high affinity for both receptors were selected. To enhance theirpotency at inhibiting VEGF-mediated processes we affinity-matured thesescVEGF proteins against VEGFR2.

To evaluate the inhibitory action of these variants on the induction ofangiogenesis, we examined their effect on the function of VEGF. SinceVEGF has been shown to be the central positive regulator of the earlygrowth of neovessels, and inhibition of VEGFR2 activity limits theability of most tumors to stimulate the formation of blood vessels, weexamined whether these variants could have an effect on (i) VEGF-inducedtyrosine phosphorylation of VEGFR2, (ii) VEGF-induced HUVECproliferation, and (iii) vitronectin-mediated cell adhesion. Inaddition, we determined whether there is correlation between the effectsabove and the ability of the variants to specifically bind recombinanthuman VEGFR2 and cells endogenously and over expressing the receptor.

Results

Evaluation of scVEGF as a scaffold for engineering new molecularrecognition. Our first step in evaluating the feasibility of usingscVEGF as a scaffold for protein engineering was to determine itscompatibility with yeast surface display. The gene for wild-type scVEGFwas prepared in two parts corresponding to a fragment of VEGF chain 1(amino acids E13 to D109) and a 14-amino acid linker SEQ ID NO:80(GSTSGSGKSSEGKG) followed by VEGF chain 2 (also amino acids E13 toD109). A mutant version (scVEGFmut) was prepared with four mutationscorresponding to key binding residues at one pole of the molecules:chain 1 F17A, E64A; chain 2 I46A, I83A (FIG. 1A) (note that residuenumbers used in this paper correspond to the residue numbers fromVEGF121, not positions in scVEGF). This construct should inhibit VEGFR2dimerization and activation by preventing a second VEGFR2molecule frombinding at the mutated face of the ligand (FIG. 2). The full genes forscVEGFwt and scVEGFmut were cloned into the yeast surface displayplasmid pCT and transformed to yeast strain EBY. Yeast expressing scVEGFproteins were tested for binding to VEGFR2, demonstrating that bothconstructs are capable of binding the receptor, and that scVEGFwt bindswith significantly higher affinity than scVEGFmut (FIG. 1B).

To evaluate whether any of the loops on the mutated pole of scVEGF wereamenable to saturation mutagenesis, we prepared libraries of three suchloops, shown in FIG. 1C, in which 5-8 amino acids were removed from theloop and replaced with randomized sequences of length 6-9 amino acids.Different registers of amino acids were removed for each of the 3 loops,and each library had just a single loop replaced with a new randomizedsequence. We tested each library for its tolerance to substitution bymonitoring the relative expression and the relative binding to 50 nMVEGFR2 (FIG. 11). These data showed that substitution of loop 3 (FIG.1C) residues SEQ ID NO:81 IKPHQGQ with 9 amino acids gave near wild-typebinding and expression levels. Loop 1 was the least tolerant tomutagenesis while loop 2 was moderately tolerant, whereas all registersand loop lengths tested for loop 3 showed very good expression andbinding levels relative to wild-type (FIG. 11).

Construction and screening of scVEGF libraries for dual VEGFR2- andα_(v)β₃ integrin-targeting. The loop mutagenesis studies suggested thatloop 3 in one chain could be replaced and engineered to bind a newtarget, such as α_(v)β₃ integrin, while the overall scVEGF protein couldstill retain binding to VEGFR2 at the opposing face (FIG. 2C). Tofacilitate integrin binding, we included an RGD recognition sequence andrandomized flanking residues. We made three libraries from scVEGFmut,corresponding to three positions of RGD within the loop by substitutingloop 3 (IKPHQGQ) with XXRGDXXXX, XXXRGDXXX, or XXXXRGDXX, where Xcorresponds to any amino acid. The libraries were transformed to yeastgiving 0.5-2×10⁷ transformants per library. The yeast libraries wereconcurrently tested for protein expression and binding to 100 nM VEGFR2.For all three libraries, nearly the entire expressing population alsobound VEGFR2, indicating that replacement of the loop again did notcompromise binding to VEGFR2 at the opposite pole of the ligand.

Library screening was performed by fluorescence-activated cell sorting(FACS). Yeast displaying scVEGF variants were labeled with α_(v)β₃integrin and an anti-cMyc antibody to simultaneously monitor proteinexpression via a C-terminal cMyc epitope tag. After treatment withfluorescently-labeled secondary antibodies, the yeast showing thegreatest receptor binding relative to expression were selected by FACS,propagated, and the process was repeated for multiple rounds. In sortround 1, the three yeast-displayed libraries were combined and a totalof ˜8×10⁷ cells were screened against 250 nM α_(v)β₃ integrin by FACS(FIG. 3A). In subsequent sort rounds, the concentration of integrin wasdropped and the number of yeast sorted was in at least a 10-fold excessof the remaining library diversity. In sort round 4, the yeast weresorted against 100 nM VEGFR2 (Fc-fusion) to remove a population ofproteins with weaker receptor binding (FIG. 2B). The final sort, round7, was performed with 25 nM α_(v)β₃ integrin (FIG. 3C).

Sixteen clones were sequenced after the seventh sort round and 7 uniquesequences were obtained (Table 2). These bi-specific clones will bereferred to as the scVEGFrgd series. Surprisingly, one of the sequenceshad a loop that was 11 amino acids long, two residues longer than the9-amino acid RGD loop we used for the library. The RGD consensus wasfound in the middle of the loop for all 7 sequences, and there was noconsensus amongst the other residues save the presence of a Pro inposition one for 5 out of the 7 sequences.

TABLE 2 Sequences of selected scVEGFrgd clones.The RGD loop only is shown (replacing residues I83-Q89 from VEGF).SEQ ID NO: 69, 7I PSVRRGDSPAS SEQ ID NO: 70, 7K PTTRGDCPDSEQ ID NO: 71, 7H PGGRGDSAY SEQ ID NO: 72, 7B PHDRGDAGVSEQ ID NO: 73, 7F STDRGDASA SEQ ID NO: 74, 7G ASGRGDGGVSEQ ID NO: 75, 7P PASRGDSPP

Affinity maturation of scVEGF mutants against VEGFR2. With scVEGFproteins capable of targeting both VEGFR2 and α_(v)β₃ integrin prepared,we next sought to improve the affinity of these variants for VEGFR2. Themutations in scVEGFmut and scVEGFrgd clones appear to decrease theiraffinities for VEGFR2, presumably a result of decreased avidity andincreased off-rate, due to their inability to bind to two VEGFR2molecules. The diminished affinities of these variants for VEGFR2relative to wild-type would presumably lead to low potency in inhibitingVEGF-mediated processes, which was observed in the previous study onsingle-chain VEGF variants. Therefore, improvement of VEGFR2 affinitiesis a critical step in preparing efficacious antagonists.

Our strategy to affinity mature the scVEGF variants against VEGFR2involved screening random mutagenesis libraries against VEGFR2 andisolating the mutants with highest affinity in increasingly stringentsorts. However, during this process it was critical that the isolatedvariants did not recover the second VEGFR2 binding site (i.e. change themutated residues back to wild-type amino acids or acquire compensatorymutations), as the resulting proteins would presumably be agonists ofthe receptor. Since we could not control for reversion mutations in ourerror-prone mutagenesis, we adopted a strategy involving screening twolibraries starting from two different scVEGF variants: scVEGFmut andscVEGFrgd-7B. Since the primary VEGFR2 binding loop in scVEGFrgd-7B wassubstituted, it would be highly unlikely that variants that recoveredbinding to VEGFR2 at this site would be isolated. Thus, if variants withcompensatory mutations were isolated from the scVEGFmut library,mutations that improve affinity at the proper VEGFR2-binding pole couldstill be identified by comparison to mutations selected in thescVEGFrgd-7B library. In addition, if similar mutations were identifiedfrom both libraries, it would serve as confirmation that those mutationswere involved in improving VEGFR2 affinity.

We prepared libraries from scVEGFmut and scVEGFrgd-7B by error-prone PCRusing nucleotide analogs and varying the mutation frequency from ˜0.2%to ˜2%. The libraries were sorted by FACS over 6 rounds using increasingstringency (lower concentrations of VEGFR2 extracellular domain). Thetotal DNA from sort rounds 5 and 6 was subjected to and additional roundof mutagenesis, then subjected to 6 rounds of FACS (FIG. 4). The finalsort round for these libraries was performed against 200 pM VEGFR2, aconcentration at which yeast-displayed scVEGFmut and scVEGFwt did notappreciably bound to the receptor. Sequencing of 14 clones selected fromthe final sorting round for the scVEGFmut and scVEGFrgd-7B librariesyielded 9 and 8 unique sequences, respectively. Protein sequences areprovided in the SEQLIST.

Most of the most common mutations, chain 1 F36L, E44G, H86Y, Q87R, Q89H,and chain 2 D63N/H are located in the correct binding interface for thepole of scVEGFmut that can bind to VEGFR2 (H86Y, Q87R, and Q89H are inthe main recognition loop) (FIG. 5). Some of the other mutationsobserved, such as chain 2 R82G and I91T, may contribute to reversion ofbinding at the opposite pole, though none of the mutations appeared inmore than one clone from both libraries. Thus these mutations are likelyto be incidental or backbone mutations that have little effect on VEGFR2binding. All of the individual clones from the scVEGFmut andscVEGFrgd-7B libraries (Table 3) tested for binding to VEGFR2 showedimproved binding for the receptor relative to scVEGFmut and scVEGFwt(FIG. 12).

TABLE 3 Clones from scVEGF affinity maturation libraries. Mutationslisted refer to positions in VEGF121 and are in addition to the specificmutations that block binding at one pole (chain 1 F17A, E64A; chain 2I46A, I83A) or substitution with the RGD loop for the scVEGFrgd-7Bclones. Clone Chain 1 mutations Chain 2 mutations scVEGFmut-A V15A,R23K, F36L, E44G, E42K, M81T H86Y scVEGFmut-E F36L, E44G, D63G, Q87RK16R scVEGFmut-J M18R, F36L, H86Y, Q89H I91T scVEGFmut-M Q22R, L32S,F47L, I76T, P53S, M78V, R82G H86Y, Q87R scVEGFrgd-7B-A F36L, H86Y, Q87R,D63N K107R scVEGFrgd-7B-C R23G, F36L, H86Y, D109G Q79R, K108RscVEGFrgd-7B-K F36L, H86Y K16R, K108E

Recombinant production of soluble scVEGF in P. pastoris. The genes forscVEGFwt, scVEGFmut, scVEGFrgd clones 7B, 7H, 7I, and 7P, as well asaffinity matured scVEGFmut clones mA, mE, mJ, and mM were cloned intothe pPIC9K vector for expression in P. pastoris strain GS115. Theproteins were expressed with a C-terminal hexahistidine tag, and eitherwith or without an N-terminal FLAG epitope tag (the untagged version isshown in the sequence listing). We originally prepared the proteins onlywith the His tag, but we discovered this tag could not be effectivelyused in cell binding assays, so we made future protein preparations thatalso included the N-terminal FLAG tag, which proved more effective formonitoring cell binding. The crude P. pastoris supernatants werepurified by Ni-NTA beads. A reduced gel of the resulting proteins showed3 bands, presumably resulting from 3 different glycosylation states andconsistent with the presence of 2 N-linked yeast glycosylation sites. Inaddition, a non-reduced gel of the resulting proteins revealed thepresence of a mix of disulfide-linked multimers. To completepurification, the proteins were first treated with endoHf to cleaveglycosylation and were then subjected to gel filtration FPLC to removethe endoHf and multimeric proteins. The fully purified protein as asingle peak in analytical gel filtration FPLC and did not revert tomultimers. MALDI mass spectrometry revealed a broad range of peaksseveral hundred Daltons larger than the expected mass and varying byseveral hundred Daltons, presumably due to small variation in cleavageof glycans.

Binding kinetics of scVEGF variants to VEGFR2 using BIAcore. SPRanalyses confirmed that all the soluble scVEGF variants injected atincreasing concentrations specifically interacted with the recombinanthuman VEGFR-2 immobilized onto the sensor chip (FIG. 6). Non-specificbinding was estimated by applying bovine serum albumin (BSA). The bestfit was obtained using a 1:1 binding model considering all proteins asmonomeric. The affinity constants (K_(D)) as well as the kineticparameters (k_(on) and k_(off)) are shown in Table 4. All the affinitymatured proteins bound VEGFR2 better than scVEGFmut (˜2 nM vs. ˜20 nMK_(D) values). The complex dissociation (k_(off)) was slower forscVEGFwt, scVEGF-mA, mE and mJ (˜3-7×10⁻⁴ 1/sec) than for scVEGFmut andscVEGF-mM (˜1×10⁻³ 1/sec). Higher k_(on) values were observed for boththe affinity matured proteins and scVEGFwt (˜2×10⁵ 1/M×sec) as comparedto scVEGFmut (˜8×10⁴ 1/sec). It is worth noting that the regeneration ofVEGFR2 immobilized on the sensor chip could be achieved by perfusing 3MMgCl₂ in the case of scVEGFwt, scVEGFmut, scVEGF-mE,mM but not forscVEGF-mA and mJ, where 3M MgCl₂ with 10 mM NaOH was needed which mightindicate different binding interactions.

TABLE 4 Binding kinetics of the scVEGF variants to immobilizedVEGFR-2^(a). Protein K_(D) (nM) K_(on) (1/M×s) K_(off) (1/s) scVEGFwt 4± 2     (2 ± 1) × 10⁵    (7 ± 2)) × 10⁻⁴ scVEGFmut 18 ± 1  (8.3 ± 0.5))× 10⁴ (1.46 ± 0.05) × 10⁻³ scVEGF-mA 2.1 ± 0.4 (2.4 ± 0.8)) × 10⁵  (4.9± 0.9) × 10⁻⁴ scVEGF-mE 2.8 ± 0.5 (2.2 ± 0.4)) × 10⁵  (6.1 ± 0.4) × 10⁻⁴scVEGF-mJ 1.6 ± 0.5    (2 ± 1)) × 10⁵     (3 ± 2) × 10⁻⁴ scVEGF-mM 3.8 ±0.8 (2.9 ± 0.6)) × 10⁵ (1.08 ± 0.07) × 10⁻³ ^(a)All of the numbers aredetermined by BIAcore analysis and represent the mean ± S.E. from atleast three separate determinations.

Affinities of soluble scVEGF variants determined by cell binding. Wetested the recombinant proteins for their ability to bind integrins andVEGFR2 on the surface of several cell types. We initially tested thescVEGFrgd analogs and affinity-matured scVEGF mutants, as well asscVEGFwt and scVEGFmut for binding to a K562 cell line that stablyexpresses α_(v)β₃ receptors and do not express VEGFR2. As expected,scVEGFwt, scVEGFmut and the affinity-matured scVEGF mutants did not bindthese cells, but the scVEGFrgd clones did. Clones 7H, 7I, and 7P boundwith K_(D) values ˜35 nM, while 7B had a substantially worse K_(D) of143 nM (FIG. 7A and Table 4). Among the scVEGFrgd protein variants, only7H, 7I, and 7P were used in subsequent studies. Both theaffinity-matured scVEGF and the scVEGFrgd proteins did not bind towild-type K562 cells, which express α5β1, and to K562 cells transfectedwith αvβ5 or αiibβ3 integrins indicating the specificity of thescVEGFrgd proteins to α_(v)β₃ receptors.

We next tested the binding of scVEGF variants against PAE cells (FIG. 7Band Table 5). PAE cells are a porcine aortic endothelial cell line thatendogenously expresses porcine α_(v)β₃ integrin. We found that thescVEGFrgd proteins, but not scVEGFwt, scVEGFmut or the affinity-maturedscVEGF proteins, bound with K_(D) values ˜25 nM to these cells. Next, wetested the proteins against a PAE cell line that has been stablytransfected to express human VEGFR2 (PAE/KDR, FIG. 7C and Table 5).scVEGFwt bound with a K_(D) of 10 nM, while scVEGFmut and the scVEGFrgdclones 7H, 7I, and 7P bound with K_(D)'s of 16-21 nM. Since binding ofthese clones to PAE/KDR cells is dependent on binding to VEGFR2, it isunsurprising that scVEGFmut and the RGD-containing clones all bind withthe same affinities and slightly less than the scVEGFwt. Importantly,the affinity-matured scVEGF mutants scVEGF-mA, -mE, and -mJ bound withsingle-digit nM affinities (similar to scVEGFwt), while scVEGF-mM hadslightly worse binding than scVEGFmut at 34 nM. The maximum bindinglevels were substantially higher for the highest affinity clonesscVEGF-mA, -mE, and -mJ. This could be due to much slower k_(off) ratesfor these clones (as discussed above in the Biacore data section).

TABLE 5 Summary of cell binding data for scVEGF variants. ProteinK562α_(v)β₃ PAE PAE-KDR HUVEC U87MG SVR scVEGFwt a a 9.8 28 ± 4 a 32 ± 5scVEGFmut a a 17 ± 8  101 ± 7  a a scVEGFrgd-7B 140 ± 10 b b b b bscVEGFrgd-7H  36 ± 12 26.5 16 ± 8  12 ± 4 15.6 18 ± 4 scVEGFrgd-7I 34 ±2 18.6 20 ± 11  6 ± 3 13.9 37 ± 5 scVEGFrgd-7P 37 ± 6 26.9 21 ± 10 45 ±5 29 31 ± 5 scVEGF-mA a a 5.7 ± 0.5 36 ± 5 a 13 ± 3 scVEGF-mE a a 6.6 ±1.8 53 ± 6 a  8 ± 4 scVEGF-mJ a a 6.9 ± 1.5 39 ± 6 a  3 ± 1 scVEGF-mM aa 34 ± 18 a a a K_(D) values are expressed in nM. a No binding wasobserved at the highest concentration tested (1 μM) b Not tested

All proteins, except scVEGFmut and scVEGF-mM, bound human umbilical veinendothelial cells (HUVEC; which express both VEGFR2 and α_(v)β₃integrin), with K_(D)s below 100 nM (FIG. 7D and Table 5). The scVEGFrgdproteins 7H and 7I, showed the highest affinity (12 nM and 6 nM,respectively), which was stronger than for the scVEGFwt, in agreementwith their ability to bind both receptors endogenously expressed onthese cells. The affinity-matured scVEGF proteins bound with a K_(D) of˜40 nM which is stronger than the binding of the scVEGFmut.

Like in HUVEC, all proteins except scVEGFmut and scVEGF-mM, bound SVRangiosarcoma cells with K_(D)s below 40 nM (FIG. 7F and Table 5).However, the affinity-matured scVEGF mutants (K_(D)'s of 8 nM) boundbetter than the scVEGFrgd proteins (K_(D)'s of 30 nM). Both HUVEC andSVR cells express VEGFR2 and α_(v)β₃ integrin, except that SVR aremurine cell lines.

As shown in FIG. 7E, all proteins bound U87MG human glioblastoma cellssimilar to PAE cells; scVEGFrgd proteins, but not scVEGFwt, scVEGFmut orthe affinity-matured scVEGF proteins, bound with K_(D) values ˜20 nM tothese cells. This is probably because the U87MG cell lines, similar toPAE cells, do not express VEGFR2 (confirmed by flow cytometryexperiments).

It is also worth noting that binding for all concentrations of thescVEGF proteins was performed in non-ligand-depleting conditions, so theaffinities are the same as what is reported here.

Inhibition of VEGF-mediated VEGFR-2 autophosphorylation in endothelialcells. The scVEGF variants were tested for their ability to inhibitVEGF-induced autophosphorylation of VEGFR-2 in HUVEC in the presence orabsence of adhesive vitronectin (FIG. 8). When coated on surfaces, verylow concentrations of vitronectin promote endothelial cell attachmentand induce spreading and migration of cells. While in the absence ofvitronectin, the bispecific scVEGFrgd variants were only slightly morepotent than the scVEGFmut (˜20% inhibition) (FIGS. 8A and C), in thepresence of vitronectin they were significantly more potent withscVEGFrgd-7I being the most active (80% inhibition) (FIGS. 8B and E).This difference is due to the ability of the bispecific variants, butnot the scVEGFmut, to block both the binding of vitronectin and VEGF toαVβ3 integrin and VEGFR2, respectively. Because of their specificity toonly VEGFR2, the ability of the affinity matured variants to inhibitVEGF-induced VEGFR2 autophosphorylation in HUVEC cells was tested onlyin the absence of vitronectin. The ability was highly correlated withthe affinity of the proteins to the immobilized VEGFR2 (BIAcore data)and to HUVEC cells. The variants with high affinity (scVEGF-mA,mE andmJ) demonstrated a strong inhibition activity, whereas the variants withlower affinity (scVEGFmut and scVEGFmM) were less active (FIGS. 8C andD). Not surprisingly, scVEGFwt did not inhibit VEGFR2 phosphorylation,and in fact it was able to promote phosphorylation when added at thehighest concentration.

Inhibition of VEGF-mediated proliferation of endothelial cells. VEGFinduced signal transduction for the proliferation of endothelial cellsis mainly mediated by VEGFR2. Therefore, we next wanted to evaluate therelationship of the abilities of the scVEGF variants to inhibitVEGF-mediated VEGFR2 autophosphorylation and endothelial cellsproliferation. The effects of the scVEGF variants on endothelialproliferation were assessed in HUVEC cells stimulated with VEGF, in thepresence or absence of vitronectin, using the DNA synthesis rate as ameasure of cell proliferation. All the proteins inhibited theproliferation of HUVECs in a dose-dependent manner.

As expected, in the absence of vitronectin, the bispecific scVEGFrgdvariants were as active as the scVEGFmut in inhibiting proliferationshowing the highest activity between 50-100 nM (FIG. 9A). In contrast,in the presence of vitronectin the bispecific variants were much moreactive (FIG. 9C). The ability of the affinity matured variants toinhibit VEGF-induced HUVEC proliferation was highly correlated with theaffinity of the proteins to the immobilized VEGFR2 receptor (BIAcoredata) and to HUVEC cells. The variants with high affinity (scVEGF-mA,mEand mJ) demonstrated a strong inhibition activity, whereas the variantswith lower affinity (scVEGFmut and scVEGFmM) were less active (FIGS. 9Band D).

Bispecific variants with high affinity for both αVβ3 integrin and VEGFR2could significantly inhibit HUVEC proliferation in the presence ofvitronectin (FIG. 9C), whereas the affinity matured mutants with highaffinity only to VEGFR2 facilitated strong inhibition of proliferationin the absence of vitronectin and weak inhibition in the presence ofvitronectin (FIGS. 9B and D, respectively). These results suggest thatthe affinity strength of the scVEGF variants mostly correlates withtheir ability to inhibit VEGF-mediated proliferation of HUVEC. As in thephosphorylation assay, scVEGFwt did not inhibit HUVEC proliferation, andin the presence of vitronectin it was actually able to promoteproliferation when added at 10-100 nM.

Inhibition of vitronectin-mediated cell adhesion by scVEGF variants. Wenext tested whether the engineered scVEGF variants could inhibit celladhesion mediated by vitronectin, the primary ligand for αvβ3 integrin.We incubated HUVEC cells with varying concentrations of proteins in96-well plate pre-coated with vitronectin to determine the ability ofthe proteins to inhibit cell adhesion. The bispecific scVEGFrgd variantswere able to block vitronectin-mediated adhesion of the HUVEC cells withIC₅₀ values<10 nM (FIG. 10). The IC₅₀ values for inhibition of celladhesion by the scVEGFmut could not be determined since there was noinhibition at the concentrations tested. scVEGFwt was able to stimulatecell adhesion to vitronectin with saturation at 60 nM.

Materials and Methods

Preparation of scVEGF Constructs and Libraries. The scVEGF Constructswere prepared by PCR assembly using overlapping primers to prepare twoinserts for chain 1 and a 14-amino acid linker/chain 2. Amplificationwas performed using end primers with NheI and BamHI restriction sitesfor chain 1 and BamHI and Mlu restriction sites for the 14-amino acidlinker/chain 2. The two inserts, followed by a cMyc epitope tag and stopcodons flanked by a XhoI restriction site were cloned into the pCT yeastdisplay vector using a multi-step cloning procedure.

Libraries were prepared starting with the scVEGFmut construct. Fullgenes with appropriate loops replaced with NNS degenerate codons wereprepared for replacement of chain 1 (loop 1) or chain 2 (loops 2 and 3).pCT vector digested with NheI/BamHI (loop 1) or BamHI/MluI (loops 2 and3) was co-electroporated with each insert into freshly preparedelectrocompetant yeast strain EBY100. The yeast were allowed to recoverfor 1 h in YPD at 30° C. then were transferred to selective SD-CAAmedia. Libraries containing the RGD sequence and randomized flankingresidues in loop 3 were prepared analogously.

Random mutagenesis libraries were prepared from scVEGFmut usingerror-prone PCR as described. Briefly, PCR was performed in the presenceof nucleotide analogs dPTP and 8-oxo-dGTP, using primers flanking thegene. The concentration of nucleotide analogs and number of cycles wasvaried in order to give a range of mutation frequencies of ˜0.2-2%. Theresulting inserts were amplified and transformed into yeast withdigested plasmid as described above. After 6 rounds of sorting, libraryplasmid DNA was extracted from yeast using a ZymoPrep kit (ZymoResearch) and subjected to error-prone PCR as described above. Thesecond generation library was similarly transformed to yeast and sortedas described below. For all libraries, transformation frequency wasestimated by dilution plating on selective SD-CAA plates. Typicallibrary sizes were 0.5-2×10⁷ transformants. For sequencing of individualclones, plasmid DNA prepared by ZymoPrep was transformed to E. coli XL-1Blue (Strategene) and individual colonies were submitted for sequencing(MCLabs, S. San Francisco, Calif.).

Sorting RGD loop libraries. Yeast displayed libraries were induced forexpression in SG-CAA media. Approximately 5-20×10⁶ yeast were labeledwith α_(v)β₃ integrin (R&D systems, octyl-glucopyranoside formulation)and a 1:200 dilution of chicken anti-cMyc antibody (Invitrogen) inintegrin binding buffer (IBB, 20 mM Tris pH 7.5, 100 mM NaCl, 1 mMMgCl₂, 1 mM MnCl₂, 2 mM CaCl₂, and 1 mg/mL BSA) for 2 h at roomtemperature. The cells were spun down, aspirated, and resuspended inice-cold BPBS (PBS+1 mg/mL BSA) containing a 1:25 dilution offluorescein-labeled mouse anti-α_(v)β₃ (BioLegend) and a 1:100 dilutionof phycoerythrin-conjugated anti-chicken-IgY (Santa Cruz Biotechnology).After 20 min on ice, the yeast were pelleted and the supernatant wasremoved. One intermediate sort was performed against VEGFR2-Fc; in thiscase a fluorescein mouse anti-Fc antibody (Sigma) was used for detectionof receptor binding. The yeast were then sorted using a Vantage SE/DiVaVantoo instrument (Stanford FACS Core Facility) and CellQuest software.In each sort ˜1-2% of yeast were collected, and in subsequent sorts atleast 10-fold more yeast were sorted than collected in the previousround. Concentrations of receptor used in each sort round were asfollows: sort 1-200 nM α_(v)β₃, sort 2-100 nM α_(v)β₃, sort 3-100 nMα_(v)β₃, sort 4-100 nM VEGFR2-Fc, sort 5-50 nM α_(v)β₃, sort 6-50 nMα_(v)β₃, sort 7-25 nM α_(v)β₃.

Sorting scVEGFmut affinity maturation libraries. Yeast displayedlibraries were sorted as described above using VEGFR2 extracellulardomain (Calbiochem) and a fluorescein-conjugated anti-VEGFR2 antibody(R&D Systems). Concentrations of receptor used in each sort round wereas follows: mutagenesis round 1, sort 1-100 nM, sort 2-50 nM, sort 4-25nM, sort 5-5 nM, sort 6-2 nM, mutagenesis round 2, sort 1-25 nM, sort2-5 nM, sort 3-2 nM, sort 4-1 nM, sort 5-500 pM, sort 6-200 pM.

Production and purification of proteins from P. pastoris. Proteinproduction was performed using the P. pastoris expression kit(Invitrogen). Genes for protein production were cloned between the AvrIIand MluI restriction sites in the Pichia expression plasmid pPIC9K with(or without) an N-terminal FLAG tag between the SnaBI and AvrIIrestriction sites and a C-terminal hexahistidine tag between the MluIand NotI restriction sites. Plasmid (5-10 μg) was linearized bydigestion with SacI and electroporated into freshly preparedelectrocompetent P. pastoris strain GS115. The yeast were then plated onRDB plates for recovery, and then transferred to YPD plates containing 4mg/mL geneticin for selection of multiple transformants. Individualcolonies were selected and grown in BMGY cultures overnight thentransferred to BMMY to induce protein production. The BMMY cultures weremaintained with ˜0.5% methanol over 3 days then tested for expressionusing Western Blot against their FLAG (or His) tag.

scVEGF affinity determination by BIAcore analysis. Binding of scVEGFproteins (incrementing concentrations from 0.2 nM to 200 nM) toimmobilized human VEGFR2 (>90% SDS PAGE purity, Calbiochem) was done viasurface plasmon resonance (SPR), using a BIAcore 3000 system (BIAcore,Inc., Uppsala, Sweden) as previously described with modifications. Thepurified VEGFR2 (40 μg/ml in 10 mM sodium acetate, pH 5.5) wascovalently attached via amine coupling to sensor chip CM5, according tothe instructions of the manufacturer, to 2300-2700 resonance units (RU).Bound ligand was then perfused in IBB/BSA/0.005% surfactant P20, pH 7.4at 25° C. at a flow rate of 30 μl/min. The specificity of analytebinding was analyzed by correction for non-specific binding, viaperfusion of non-coupled control channels. Association (k_(on)) anddissociation (k_(off)) rate constants were calculated via curve fitting,using the BIAevaluation 2.0 software, assuming a 1:1 model, consideringall proteins as monomeric at concentrations tested. The affinityconstant (K_(D)) was calculated from the ratio of dissociation rate(k_(off))/association rate (k_(on)). The rapid increase and decrease inresonance signal, preceding association and dissociation respectively(buffer jumps), were excluded from evaluation. The chip was regeneratedby injection of 3M MgCl₂ with 10 mM NaOH for 30 s at 30 μl/min.

Cell binding assays. Wild-type K562 cells were maintained in IMDM media(Gibco) supplemented with 10% FBS. Media for K562 cells expressingintegrins also had 1.2 mg/mL geneticin. Cells were maintained insuspension at concentrations of ˜2-20×10⁵ cells/mL. PAE and PAE/KDRcells were grown in F-12 (Ham's) Nutrient Media (Gibco) with 10% FBS and1% Pen/strep. HUVEC cells were grown in full EGM-2 media (Lonza)containing 2% FBS and growth factor supplements. U87MG cells were grownin DMEM (high glucose) media (Gibco) containing 10% FBS and 1%Pen/strep. SVR cells were grown in DMEM media containing 10% FBS and 1%Pen/strep. Cells grown on plates were split at 80-90% confluence using0.05% trypsin-EDTA.

For cell binding assays, 10⁵ cells were used per condition. Cells weresuspended in IBB (0.1-1 mL volume) and protein was added as a 10× or100× stock in an amounts that were sufficient to avoid ligand depletionat all ligand concentrations tested. The cells were incubated at 4° C.with gentle agitation to prevent settling for 4-6 h, then spun down at1000 rpm (0.1 rcf) at 4° C. for 3 min and the supernatant was aspirated.The cells were then resuspended in 20 μL BPBS containing a 1:40 dilutionof fluorescein-conjugated anti-His antibody (for K562 cells) or a 1:100dilution of phycoerythrin-conjugated anti-FLAG antibody (for the othercells). After 20 minutes, the cells were resuspended in 1 ml BPBS,centrifuged and the supernatant was aspirated. The cells were kept aspellets on ice until analysis by flow cytometry. Mean fluorescence foreach concentration was calculated using FlowJo (Treestar, Inc) thenplotted versus log concentration, and the data were fit to a sigmoidalcurve to calculate dissociation constants using Kaleidograph (SynergySoftware).

VEGFR-2 autophosphorylation assay. VEGFR-2 phosphorylation assay wascarried out following the procedure previously described with smallmodifications. Briefly, subconfluent HUVECs were grown in growth factorand serum-depleted EBM-2 medium for 20 h prior to experimentation. Afterpretreatment with 1 mM sodium orthovanadate (Na₃VO₄) for 20 min, thecells were incubated in the presence of 1 nM of VEGF121 and differentconcentrations of the protein variants for 10 min at 37° C. The cellswere then washed in phosphate-buffered saline (PBS) with 1 mM Na₃VO₄ andlysed in ice-cold 1% Triton X-100 lysis buffer for 2 h (20 mM Tris pH7.4, 150 mM NaCl, 1% TritonX-100, 1×APC, 1×AEBSF, 1 mM Na₃VO₄, 1×complete protease inhibitor tablet). The lysates were clarified bycentrifugation (13,000 rpm for 10 min at 4° C.). Protein concentrationswere measured using a Bio-Rad protein assay and the same amounts ofprotein of each sample were used for analysis. Cell lysates weresubjected to 4-12% SDS-PAGE and transferred to a nitrocellulose sheet.The blots were incubated with a blocking solution (5% milk containingTBST washing buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.3% Tween20)) and probed with primary antibodies (Y951 or VEGFR2) diluted inblocking solution for overnight at 4° C. The signals were visualizedusing HRP-conjugated anti rabbit secondary antibodies and exchangedchemiluminescence (ECL plus, Amersham) according to the manufacturer'sinstructions. The immunoreactive bands were quantified on a chemidocsystem. Blots were stripped and re-probed to determine total amounts ofVEGFR2 present. Unstimulated (basal) and VEGF121-stimulated cells wereused as negative and positive controls, respectively. The above assaywas also done in the presence of vitronectin. Plates were coated withvitronectin as previously described with small modifications. In brief,plates were coated with 0.2 μg/cm² of vitronectin (Promega) for 2 hrs at37° C. in DPBS. Well were rinsed twice with DPBS before cell plating.

Cell proliferation assays. Proliferation was assayed as described.Briefly, HUVEC cells (4×10³ per well) were placed in 96 well plates ingrowth factor-containing EBM-2 media for overnight h at 37° C./5% CO₂.Cells were incubated in growth factor and serum-free EBM-2 medium for 20h at 37° C./5% CO₂ to suppress growth. Cells were then incubated withvarying concentrations of engineered scmVEGF proteins and 1 nM VEGF121for 48 h at 37° C./5% CO2. For the last 24 h of incubation, 1 μCi (20Ci/mmol) [³H]thymidine were added to each well in 50 μl of EBM-2 media.Plates were then frozen at −80° C. and thawed again at room temperature.[³H]thymidine incorporation was be measured by harvesting the cells ontoglass fiber filtermats using a Mach IIIM harvester and performingscintillation counting with a Wallac MicroBeta. Unstimulated andVEGF121-stimulated cells were used as negative and positive controls,respectively. The above assay was also done in the presence of Assayvitronectin as previously described. Wells were coated with vitronectinas previously described with small modifications. In brief, plates werecoated with 0.2 μg/cm² of vitronectin (Promega) for 2 hrs at 37° C. inDPBS. Well were rinsed twice with DPBS before cell plating.

Vitronectin-mediated cell adhesion assays. Assay for HUVEC adhesion tovitronectin was performed as described with small modifications. Inbrief, plates were coated with 0.2 μg/cm² of human vitronectin (Promega)for 2 hrs at 37° C. in DPBS. After two rinses with DPBS, wells wereblocked with sterile 2 mg/ml BSA for 1 hr at room temperature and rinsedtwice before cell plating. Adhesion assay was conducted as describedbefore. Briefly, varying concentrations of scVEGF proteins were mixedwith HUVEC cells and added to vitronectin-coated 96-well plates. Theplates were incubated at 37° C. with 5% CO2 for 2 hrs, then the wellswere washed two times with PBS. A solution of 0.2% crystal violet in 10%ethanol was added to the wells for 10 min, then the wells were washedthree times with PBS. Solubilization buffer (a 1:1 mixture of 0.1 MNaH₂PO₄ and ethanol) was added and the plate was gently rocked for 15min to completely solubilize the crystal violet. Absorbance of the wellswas measured at 600 nm with a microtiter plate reader (BioTekInstruments), and data were background subtracted with a negativecontrol containing no cells. IC₅₀ values were generated by fitting asigmoidal curve to plots of log concentration peptide versus percentadhesion. Data was normalized using samples containing no competingprotein. Data are presented as average values with standard deviations.Experiments were performed at least three times.

TABLE 6 Libraries used for testing VEGF loop tolerance. Loop 1A: ΔNDAGLE(Replace with loop sizes 6, 7, 8, 9) Loop 1B: ΔNDAGL (Replace with loopsizes 6, 7, 8, 9) Loop 2A: ΔYPDEIEYA (Replace with loop sizes 7, 8, 9)Loop 2B: ΔYPDEIEY (Replace with loop sizes 7, 8, 9) Loop 2C: ΔPDEIEYA(Replace with loop sizes 7, 8, 9) Loop 2D: ΔPDEIEY (Replace with loopsizes 7, 8, 9) Loop 2E: ΔDEIEYA (Replace with loop sizes 7, 8, 9) Loop2F: ΔDEIEY (Replace with loop sizes 7, 8, 9) Loop 3A: ΔIKPHQGQ (Replacewith loop sizes 7, 8, 9) Loop 3B: ΔIKPHQG (Replace with loop sizes 7, 8,9)Sequences

SEQ ID NO:1 shows the amino acid sequence of VEGF-121. SEQ ID NO:2 showsthe 97-amino acid core region of VEGF-121 which was used to create thesingle-chain VEGF variants of the present invention. SEQ ID NO:3 showsthe amino acid sequence of a single-chain variant of VEGF consisting oftwo identical core domains joined by a linker (MW=25249.7; ε278=13616M⁻¹cm⁻¹=0.5393 (mg/mL)⁻¹cm⁻¹). SEQ ID NO:4 shows the amino acid sequenceof a single-chain variant of VEGF with amino acid mutations that abolishbinding to VEGFR2 at one pole, but allow binding at the opposite pole(MW=25031.4, ε278=13616 M⁻¹cm⁻¹=0.5440 (mg/mL)⁻¹ cm⁻¹). SEQ ID NO:5 isscVEGF_(RGD)-7B comprising an RGD motif (MW=24927.3, ε278=12216M⁻¹cm⁻¹=0.4901 (mg/mL)⁻¹cm⁻¹). SEQ ID NO:6 is scVEGF_(RGD)-7H(MW=24883.2, ε278=13616 M⁻¹cm⁻¹=0.5472 (mg/mL)⁻¹cm⁻¹). SEQ ID NO:7 isscVEGF_(RGD)-7I (MW=25132.5, ε278=12216 M⁻¹cm⁻¹=0.4861 (mg/mL)⁻¹cm⁻¹).SEQ ID NO:8 is scVEGF_(RGD)-7P (MW=24887.2, ε278=12216 M⁻¹cm⁻¹=0.4909(mg/mL)⁻¹cm⁻¹).

SEQ ID NO: 9-18 provides the amino acid sequence of scVEGFmut affinitymatured sequences. SEQ ID NO:19-27 provides the amino acid sequences ofscVEGFrgd-7B affinity matured sequences.

Although the foregoing invention and its embodiments have been describedin some detail by way of illustration and example for purposes ofclarity of understanding, it is readily apparent to those of ordinaryskill in the art in light of the teachings of this invention thatcertain changes and modifications may be made thereto without departingfrom the spirit or scope of the appended claims. Accordingly, thepreceding merely illustrates the principles of the invention. It will beappreciated that those skilled in the art will be able to devise variousarrangements which, although not explicitly described or shown herein,embody the principles of the invention and are included within itsspirit and scope.

What is claimed is:
 1. A human vascular endothelial growth factor (VEGF)variant polypeptide, comprising: a first human VEGF polypeptidecomprising F17A and E64A substitutions, and linked through a polypeptidelinker to a second human VEGF polypeptide comprising I46A mutation,wherein said second human VEGF polypeptide comprises anintegrin-recognition RGD motif containing loop selected from thesequences set forth in any one of SEQ ID NO:29-SEQ ID NO:75 andreplacing a loop 3 sequence of said VEGF polypeptide to provide asingle-chain VEGF antagonist, whereby the VEGF variant exhibits a highaffinity to VEGF receptor-2 (VEGFR2) and alpha v beta 3 integrin.
 2. TheVEGF variant of claim 1, wherein said loop 3 sequence is I83-Q89.
 3. AVEGF variant, having an amino acid sequence set forth in any one of SEQID NO:5-8, and SEQ ID NO:19-27.
 4. A VEGF variant, having an amino acidsequence set forth in SEQ ID NO:9-18 and 19-27, further comprising themodification of replacing amino acid residues of loop 3 in one of saidhuman VEGF with an RGD motif.
 5. A single-chain antagonistic human VEGFvariant having an amino acid sequence set forth in SEQ ID NO:9-18.
 6. AVEGF variant according to any one of claims 1, 4 or 5, furthercomprising a functional conjugate.
 7. The VEGF variant according toclaim 6, wherein said functional conjugate is a detectable moiety.
 8. Apharmaceutical composition comprising a VEGF variant according to anyone of claims 1, 4 or 5, and a pharmaceutically acceptable excipient. 9.An isolated nucleic acid encoding a VEGF variant according to claim 5.10. A human vascular endothelial growth factor (VEGF) variantpolypeptide, comprising: a first human VEGF polypeptide linked through apolypeptide linker to a second human VEGF polypeptide: wherein saidfirst human VEGF polypeptide comprises F17A and E64A amino acidsubstitutions; wherein said second human VEGF polypeptide comprises I46Aand I83A amino acid substitutions, and an integrin-recognition RGD motifcontaining loop having the sequence set forth in SEQ ID NO:69, toprovide a single-chain VEGF antagonist that exhibits a high affinity toVEGF receptor-2 (VEGFR2) and alpha v beta 3 integrin.
 11. A method ofinhibiting VEGF-mediated proliferation or migration of endothelialcells, the method comprising contacting an endothelial cell with a VEGFvariant according to claim
 5. 12. The method of claim 11 wherein saidcontacting is performed in vivo.